Sepsis is a potentially lethal bacterial infection that commonly results in the impairment of multiple organ systems [1]. The kidney is an organ that exhibits a high susceptibility to sepsis-evoked damage at an earlier stage [2]. Severe renal injury, characterized by significant damage to the glomeruli, tubules, or renal interstitium, leads to acute renal failure and is strongly associated with the survival rate and prognosis of patients with sepsis. Renal injury is one of the independent risk factors for mortality in septic patients [3]. Prompt identification and early management to prevent renal injury are essential. However, clinically effective renal-protecting medications are currently limited, making it imperative to explore more effective drugs and techniques.

Lipopolysaccharide (LPS), also known as endotoxin, is a crucial virulence factor found in the outer membrane of gram-negative bacteria. It plays a significant role in triggering systemic inflammatory response syndromes and acute renal injury in sepsis [4]. The LPS-induced renal injury model in mice is a widely employed animal model utilized for investigating the mechanisms and potential treatments for renal injury associated with sepsis [5]. In our study, a LPS-induced renal injury model was utilized to simulate the circumstances of renal damage in human infection or inflammatory conditions, to further elucidate the pathogenesis of sepsis-related renal damage, and to explore potential renal protection medications and methods.

Oligomeric proanthocyanidin (OPC), a naturally occurring antioxidant, was initially discovered in pine bark and subsequently proven to be prevalent in various plants, including grape, ginkgo biloba, rhubarb, and hawthorn, especially in grape seed extracts [6]. Pycnogenol, a pine bark extract packed with OPC, emerged as a popular nutritional supplement approved for commercialization in Asia-Pacific due to its unique antioxidant properties [7]. Modern pharmacology shows that OPC possesses various beneficial biological properties, including antioxidant, anti-inflammatory, anticancer, hypoglycemic, and cardiovascular protective effects [8-10]. OPC has significant antibacterial activity, inhibiting the growth of Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Bacillus cereus, and Candida albicans, along with the inflammatory response triggered by them [9]. Additionally, numerous studies present evidence that OPC positively guards against a wide range of inflammatory disorders, including mastitis, gastroenteritis, tracheitis, and arthritis [11-14]. Nevertheless, the significance of OPC in sepsis-induced acute renal failure remains unclear. We discovered that OPC is capable of minimizing tubular damage driven by sepsis and may be a viable therapy for protecting the kidneys of septic patients. However, the mechanism remains unknown. Our study is aimed at examining the precise mechanism by which OPC provides protection to the kidney against LPS-induced renal tubular injury and identifying potential therapeutic targets and strategies for managing sepsis-induced renal injury.

Agents

OPC (Item Number: HY-N0794), LPS (Item Number: HY-D1056), and ATP (Item Number: HY-B2176) were acquired from MedChemExpress Co., LLC. MACKLIN Co., LLC provided ulinastatin (item no. 80449-31-6). Antibodies against p-AKT (Ser473) (lot no. 4060), AKT (lot no. 4691), p-PI3K p85 (Tyr458)/p55 (Tyr199) (lot no. 17366), PI3K p110 (lot no. 4249), p-IKKα/β (Ser176/180) (lot no. 36214), p-NFκB (Ser536) (lot no. 3033), nuclear factor κB (NFκB) (lot no. 8242), α-Tubulin (lot no. 5335), horseradish peroxidase-conjugated anti-rabbit IgG (lot no. 7074), and horseradish peroxidase-conjugated anti-mouse IgG (lot no. 7076) were obtained from CST Shanghai Biological Reagents. Thermo Fisher Scientific supplied BI with fetal bovine serum (FBS). Kaiji Biological Co., Ltd. procured Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12).

Animals

All experiments were conducted in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals at the Experimental Animal Center of the Affiliated Hospital of Nanjing University of Chinese Medicine. The Research Ethics Committee of the Affiliated Hospital of Nanjing University of Chinese Medicine approved the protocol (file: 2023 DW-03-18). 36 C57/B6 male mice (aged 6–8 weeks and weighing 18–22 g) were obtained from the Animal Center of Yangzhou University (Jiangsu, China). Mice were adaptively fed for a week before being randomized into six groups: control (n = 6), LPS (n = 6), LPS + OPC low-concentration (2.5 mg/kg), LPS + OPC medium-concentration (5 mg/kg), LPS + OPC high-concentration (10 mg/kg), and LPS + ulinastatin (n = 6). The renal injury models were established with a single intraperitoneal injection of LPS (10 mg/kg). The OPC-treated groups received intragastric administration of varying OPC concentrations daily. The ulinastatin group received intraperitoneal administration of ulinastatin (50,000 U/kg). Three days before the model experiment, OPC and ulinastatin began to be administered. After 24 h of the model experiment, mice were anesthetized using a mouse gas animal anesthesia machine with a 5% inhalant anesthetic (isoflurane) at a flow rate of 1,000 ml/min. Blood was collected by cardiac blood sampling. Then, all of the mice were sacrificed by carbon dioxide asphyxiation, and renal tissues were collected.

Cells

The mouse tubular epithelial cells (MTECs) were obtained from ATCC and maintained in DMEM/F12 with 5% FBS. All media were supplemented with 100 mg/ml streptomycin, 100 U/ml penicillin G, and 0.25 mg/ml amphotericin B. The cells were cultured at 37ºC with 5% CO₂.

Gene sequencing

Appropriate quantities of tissue samples were collected in tubes with corresponding numbers. The tissue cells were completely lysed by adding 1.5 ml of TRIzol lysate solution to the tissue grinder, grinding it for 30 sec, and letting it sit for 5 min. Under the protection of liquid nitrogen, sufficient quantities of tissue samples were ground into powder and transferred to lysis solutions. Allow 5 min for adequate lysis of tissue cells by lying supine. The samples of ground and fragmented tissue were centrifuged at 12,000 g for 5 min at 4°C. The supernatant was transferred to an EP tube containing 300 L of a 24:1 mixture of chloroform and isoamyl alcohol, then vigorously mixed by inversion and centrifuged at 12,000 g for 8 min. Draw the supernatant into a new 1.5 ml centrifuge tube, add 2/3 volume of isopropanol to the supernatant, gently invert and mix well, and then place the tube in a –20°C refrigerator for at least 2 h. Centrifuge at 17,500 g for 25 min at 4°C, discard the supernatant, wash with 0.9 ml of 75% ethanol, suspend the particle upside down, and centrifuge at 17,500 g for 3 min at 4°C. After a brief centrifugation, the supernatant was discarded, and the remaining liquid was aspirated and desiccated for 3–5 min. Dissolve the precipitate in 20–200 μl of DEPC water or water without RNase. The extracted RNA samples were sent to BGI for RNA sequencing, and based on the sequencing results, the differential genes and enriched signaling pathways were analyzed.

Renal function analysis

The serum creatine (Scr) and blood urea nitrogen (BUN) levels were determined by employing the Quantichrom Creatinine Assay Kit (Item No. DICT-500) and Quantichrom Urea Assay Kit (Item No. DIUR-100) procured from BioAssay Systems. All procedures were conducted in accordance with the manufacturer's instructions.

Inflammatory cytokines analysis

The inflammatory cytokines of blood serum and renal cortex tissues, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10), were detected using Elisa kits procured from MULTI SCIENCES (Item No. TNF-α: EK282/4-96, IL-1β: EK201B/3-96, IL-6: EK206/3-96. and IL-10: EK210/4-96). Following the preparation of renal cortical tissue into tissue homogenates, protein concentrations were determined using a BCA reagent (Item No. P0010, Beyotime). The respective kits were employed to test the separated serum and renal cortical tissue homogenates. All procedures were conducted in accordance with the manufacturer's instructions.

Detection of oxidative stress markers

Renal cortex tissues were utilized to measure superoxide dismutase (SOD) and catalase (CTA) activity, as well as glutathione (GSH) and malondialdehyde (MDA) concentrations, using the SOD assay kit (Item No. A001-3-2), CTA assay kit (Item No. A007-1-1), total GSH assay kit (Item No. A006-2-1), and MDA assay kit (Item No. A003-1-2), respectively. All procedures were carried out according to the manufacturer's instructions.

Renal histopathological analysis

The renal tissues of mice were subjected to fixation using a 4% paraformaldehyde solution. Subsequently, the tissues were embedded in paraffin, sliced into slides, and mounted onto glass slides. The slides were then deparaffinized and stained using hematoxylin and eosin (H&E) as well as Periodic Acid-Schiff (PAS) staining kits. The process of staining was carried out and subsequently observed using an inverted light microscope. The renal tubular injury was semi-quantitatively graded from 0 to 4+ based on the proportion of pathological injury area under a single field of view, as previously reported [15]: 0 indicates no anomalies. Changes impacting less than 25% of the sample are graded 1+; changes affecting 25% to 50% are graded 2+; changes affecting 50% to 75% are graded 3+; and changes affecting more than 75% are graded 4+.

Cell viability

Cells were uniformly distributed in 96-well plates and, upon reaching proper confluency, were treated with the appropriate drugs. After a period of 24 h, the cells were examined for any morphological alterations using an inverted light microscope. A solution of thiazolyl blue (MTT) with a concentration of 5 mg/ml was prepared. Following a 24-h treatment of 96-well plates, the serum-free medium was substituted, and subsequently, 10 μl of MTT solution was introduced to each well. After an incubation period of 4 h in the cell culture chamber, the culture medium was discarded. A volume of 150 microliters of dimethyl sulfoxide was introduced into each well in order to facilitate the dissolution of the purple crystals present in the wells. Following the complete dissolution of the purple crystals, the absorbance at a wavelength of 570 nm was quantified in order to determine the viability of the cells.

Hoechst 33342 / propidium iodide (PI) staining

Cells were distributed in 6-well plates along with coverslips to facilitate uniform cell growth on the coverslips. Following the completion of cell growth, an intervention was performed using LPS (5 μg/ml) along with ATP (3 mM). Following a 24-h period of stimulation, the samples were subjected to fixation using a 4% paraformaldehyde solution for a duration of 30 min, after which the coverslips were detached. The coverslips were treated with an anti-fluorescence quenching mounting solution that contained Hoechst 33342 and PI. The coverslips, along with the cells, were carefully covered to ensure maximum contact between the cells and the mounting solution while minimizing the presence of bubbles. Subsequently, the cell samples were examined using a fluorescence microscope. Apoptosis was assessed quantitatively by the ratio of the number of apoptotic cells to the total number of cells.

Western blot analysis

Cells were extracted, and membranes and nuclear membranes were lysed to release proteins. Extracted protein samples were added to polyacrylamide gel wells for electrophoretic separation. The separated proteins were transferred from the gel to the polyvinyl diol membrane by a wet transfer system. Blocking was performed using a blocking solution for 1 h. The corresponding primary antibody was added to the transfer membrane containing protein, and after overnight incubation, the membrane was washed three times with tris-buffered saline with tween-20 (TBST) for 10 min each time. The secondary antibody was added to the transfer membrane, and the membrane was washed three times again after 1 h of incubation for detection. The bands were visualized using the Chemidoc Imaging System (Tanon 4600SF). ImageJ (1.8.0) was used to quantitatively analyze the Western blot bands.

Statistical analysis

Values are analyzed and expressed as the mean minus the standard deviation. The comparisons of different groups were analyzed by the unpaired t-test using SPSS Statistics 22.0 (IBM Corp.). A p-value of less than 0.05 indicates a statistically significant difference.

OPC attenuates LPS-induced tubular injury in vivo

To determine the impact of OPC on renal injury induced by LPS, OPC was administered in vivo as a therapeutic intervention following LPS administration to mice. Fig. 1A, B depict a significant reduction in the levels of Scr and BUN induced by LPS after administration of OPC. Notably, the observed improvements were more pronounced than those of a positive control (ulinastatin), which has been reported to ameliorate LPS-induced renal injury. The LPS group exhibited significant tubular epithelial cell enlargement and shedding, a decrease in brush border microvilli, luminal narrowing and occlusion, and other detrimental alterations, as revealed by H&E and PAS staining of renal tissues. Meanwhile, OPC significantly ameliorated these detrimental alterations (Fig. 1C). N-gal serves as a significant indicator of acute tubular injury. The renal cortex of mice in the LPS group exhibited a noteworthy increase in N-gal expression, as depicted in Fig. 1D, E. Conversely, OPC demonstrated a significant ability to inhibit this alteration. All outcomes demonstrated that OPC exhibited the capacity to mitigate tubular injury induced by LPS in vivo.

Figure 1. Effects of OPC on LPS-induced renal tubular injury in vivo. (A, B) The changes in serum renal function test parameters. OPC (2.5, 5, 10 mg/kg) and ulinastatin (50,000 U/kg) treatments were started 3 days before LPS (10 mg/kg) injection and terminated after the administration of LPS. Blood and renal tissues were collected 24 h after LPS injection (n = 6). (C) Renal histological changes were observed using H&E and PAS staining, showing tubules of the indicated groups. Bar = 200 μm. (D) The renal tubular injury semi-quantitatively scored by histopathologist according to the standards as described above (n = 6). (E) The role of OPC on the expression of N-gal in the kidney. Sample lysates from representative mice renal cortices of the control, LPS, and LPS + OPC 10 mg/kg groups were analyzed using Western blotting (n = 3). (F) Semi-quantitative densitometric analysis of D for N-gal. The p-values are indicated at the top of the bars. OPC, oligomeric proanthocyanidin; LPS, lipopolysaccharide; H&E, hematoxylin and eosin; PAS, Periodic Acid-Schiff; BUN, blood urea nitrogen; Ctrl, control. ^####p< 0.0001; ^##p < 0.01 versus control. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 versus LPS, respectively.

OPC attenuates LPS-induced tubular injury in vitro

To further assess the effect of OPC on renal tubules, we administered OPC to renal tubular epithelial cells in vitro. As shown in Fig. 2A, B, LPS induced atypical morphology, detachment, fragmentation, and a decrease in tubular epithelial cell viability. In contrast, the application of OPC resulted in a significant reduction of these deleterious changes. Fig. 2C demonstrated that OPC significantly reduced LPS-induced apoptosis in cells. All results indicated that OPC had the capacity to mitigate LPS-induced tubular epithelial cell injury in vitro.

Figure 2. Effects of OPC on LPS-induced tubular injury in vitro. (A) The role of OPC on morphological changes of mouse tubular epithelial cells (MTEC) triggered by LPS. MTEC cells were incubated with OPC (5 μM) for 1 h and challenged with LPS (5 μg/ml) and ATP (3 mM) for 1 day. Changes in cell morphology were determined using an inverted microscope (100×). Bar = 400 μm. (B) Effects of OPC on cell viability. Cell viability was tested using the MTT assay (n = 3). (C) Apoptotic staining of MTEC cells. MTEC cells were treated with OPC (5 μM) in a 6-well plate for 1 h and then incubated with LPS (5 μg/ml) + ATP (3 mM) for 1 day. Then, the apoptotic cells were determined using the Hoechst 33342 / PI staining. (D) Percentage of apoptotic cells. The p-values are indicated at the top of the bars. OPC, oligomeric proanthocyanidin; LPS, lipopolysaccharide; PI, propidium iodide; Ctrl, control. ^###p < 0.001, ^####p < 0.0001 versus control. ***p < 0.001, ****p < 0.0001 versus LPS.

Analysis of potential mechanisms of OPC against LPS-induced renal injury by RNA sequencing

We sequenced the RNA of renal cortical tissue from indicated mice to obtain a deeper understanding of the putative mechanism by which OPC mitigates tubular injury induced by LPS. Fig. 3A, B revealed that a total of 6,491 genes exhibited differential expression in mice treated with LPS as compared to mice in a normal state. Among these genes, 3,224 were observed to be up-regulated, while 3,267 genes were down-regulated. In contrast to the LPS group, a total of 4,953 genes exhibited differential expression in the renal cortex of LPS mice treated with OPC. Among these genes, 2,573 were found to be up-regulated while 2,380 were down-regulated (Fig. 3C, D). A comprehensive analysis was conducted to compare the differential genes, resulting in the identification of a shared set of 3,465 genes that exhibited alterations in the renal tissue of mice treated with OPC in both the normal and LPS groups (Fig. 3E, F). The enrichment analysis of the KEGG signaling pathway for these genes demonstrated that the potential mechanism of OPC in mitigating LPS-induced renal damage primarily involves the enrichment of reactive oxygen species (ROS), oxidative phosphorylation, thermogenesis, and other signaling pathways (Fig. 3G). In conjunction with pertinent literature and established fundamental discoveries, it is postulated that oxidative stress, inflammatory response, PI3K/AKT, and NFκB signaling pathways contribute significantly to the tubular injury induced by LPS, as well as to the mechanism through which OPC exerts its protective influence.

Figure 3. RNA sequencing analysis of differential genes in mice. (A) Scatter plot of differential gene distribution in the kidney among the control and LPS groups. (B) Histogram showing the number of up- and down-regulated differential genes in the kidney among the control and LPS groups. (C) Scatter plot of differential gene distribution in the kidney among the LPS and OPC-treated groups. (D) Histogram showing the number of up- and down-regulated differential genes in the kidney among the LPS- and OPC-treated groups. (E) Heat map showing differential gene distribution in kidney from the control, LPS, and OPC-treated groups. (F) Wenckebach plots showing the number of common differential genes before and after OPC treatment in the LPS model. (G) Bubble plots depicting KEGG signaling network enrichment of differential genes before and after OPC treatment in the LPS model. LPS, lipopolysaccharide; OPC, oligomeric proanthocyanidin; KEGG, Kyoto Encyclopedia of Genes and Genomes; Ctrl, control.

OPC attenuates LPS-induced oxidative stress injury in renal injury

To evaluate the impact of oxidative stress on LPS-induced renal injury, we conducted measurements of GSH content, SOD activity, CTA activity, and MDA level in the renal cortex of mice. Results revealed that in the renal cortex of mice, the administration of LPS led to a reduction in GSH content, SOD and CTA activities, and an increase in MDA content. However, OPC displayed a significant ability to prevent these alterations (Fig. 4). The findings indicate that OPC has the potential to mitigate the oxidative stress injury induced by LPS in the kidney.

Figure 4. Effects of OPC on LPS-induced oxidative stress injury in the kidney. (A) The content of GSH in renal cortical tissues of the indicated groups (n = 5). (B) The SOD activity in renal cortical tissues of the indicated groups (n = 5). (C) The CTA activity in renal cortical tissues of the indicated groups (n = 5). (D) The content of MDA in renal cortical tissues of the indicated groups (n = 5). The p-values are indicated at the top of the bars. OPC, oligomeric proanthocyanidin; LPS, lipopolysaccharide; GSH, glutathione; SOD, superoxide dismutase; CTA, catalase; MDA, malondialdehyde; ns, not statistically. ^#p < 0.05, ^##p < 0.01 versus control. *p < 0.05, **p < 0.01, and ***p < 0.001 versus LPS, respectively.

OPC reduces LPS-induced inflammation in renal injury

The inflammatory response serves as a crucial foundation for the development of renal damage induced by LPS. As shown in Fig. 5, LPS resulted in a notable inflammatory reaction in mice. Specifically, the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were significantly elevated in both serum and renal cortical tissue in the LPS group. Conversely, the anti-inflammatory factor IL-10 exhibited a significant decrease in the LPS group. However, these alterations were effectively reversed following treatment with OPC. All outcomes indicate that OPC exhibits the capacity to suppress inflammatory responses induced by LPS in renal injury.

Figure 5. Effects of OPC on LPS-induced inflammation in vivo. (A–D) The levels of IL-1β, IL-6, TNF-α, and IL-10 in blood serum from mice. (E–H) The levels of IL-1β, IL-6, TNF-α, and IL-10 in renal tissues from mice. The p-values are indicated at the top of the bars. OPC, oligomeric proanthocyanidin; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-alpha; IL-1β, interleukin-1 beta; IL-6, interleukin-6; IL-10, interleukin-10, ns, not statistically. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 versus control. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 versus LPS, respectively.

OPC activates the PI3K/AKT signaling pathway and inhibits the NFκB signaling pathway against LPS-induced renal tubular injury

RNA sequencing analysis indicates that the PI3K-AKT and NFκB signaling pathways are significantly involved in the development of tubular injury induced by LPS. To conduct a more comprehensive examination of the involvement of PI3K-AKT and NFκB signaling pathways in the improvement of OPC against LPS-induced renal injury, we employed OPC as an intervention in vitro. As shown in Fig. 6A, B, the expression of p-PI3K and p-AKT were reduced, whereas the levels of p-IKKα/β and p-NFκB were elevated in tubular epithelial cells stimulated with LPS, and these changes occurred in a time-dependent manner (Fig. 6E, F). These results indicate that LPS suppressed the PI3K-AKT pathway and activated the NFκB signaling pathway. However, after intervention with OPC, these alterations were reversed (Fig. 6C, D, G, and H), suggesting that the protective effects of OPC on tubular cells could be mediated through the modulation of the PI3K-AKT and NFκB signaling pathways.

Figure 6. Role of OPC in the PI3K/AKT and NF κB signaling pathways against LPS-induced renal tubular injury. (A) Expression of PI3K/AKT signaling pathways in LPS-induced renal tubular injury. Mouse tubular epithelial cells (MTEC) were treated with LPS (5 μg/ml) + ATP (3 mM) in a time-dependent manner. Cell lysates were obtained for Western blotting analysis of p-PI3K, PI3K, p-AKT, and AKT. (B) Analyzing A for a semi-quantitative densitometric ratio of p-PI3K to total-PI3K and p-AKT to total-AKT (n = 3). (C) The influence of OPC on the expression of the PI3K/AKT signaling pathway in MTEC cells, MTEC cells were treated with OPC (5 μM) for 1 h before being exposed to LPS (5 μg/ml) + ATP (3 mM) for 24 h. Cell lysates were obtained for the Western blotting analysis of p-PI3K, PI3K, p-AKT, and AKT. (D) Analyzing C for a semi-quantitative densitometric ratio of p-PI3K to total-PI3K and p-AKT to total-AKT (n = 3). (E) Expression of NFκB signaling pathway induced by LPS In a 6-well plate, MTEC cells were treated with LPS (5 μg/ml) + ATP (3 mM) in a time-dependent manner. Cell lysates were obtained for Western blotting analysis of p-IKKα/β, p-NFκB, NFκB, and GAPDH. (F) Analyzing E for a semi-quantitative densitometric ratio of p-NFκB to total-NFκB and p-IKKα/β to GAPDH (n = 3). (G) The influence of OPC on the expression of NFκB signaling pathway in MTEC cells. MTEC cells were treated with OPC (5 μM) for 1 h before being exposed to LPS (5 μg/ml) + ATP (3 mM) for 24 h. Cell lysates were obtained for the Western blotting analysis of p-IKKα/β, p-NFκB, NFκB, and GAPDH. (H) Analyzing G for a semi-quantitative densitometric ratio of p-NFκB to total-NFκB and p-IKKα/β to GAPDH (n = 3). The p-values are indicated at the top of the bars. OPC, oligomeric proanthocyanidin; LPS, lipopolysaccharide; NFκB, nuclear factor κB; Ctrl, control. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 versus control. *p < 0.05 and **p < 0.01 versus LPS, respectively.

The current study demonstrated that OPC exhibits the capacity to alleviate renal tubular injury induced by LPS through its anti-oxidative and anti-inflammatory properties, as well as its activation of the PI3K/AKT signaling pathway and inhibition of the NFκB signaling pathway. The PI3K/AKT and NFκB signaling pathways play a crucial role in the pathogenesis of sepsis-associated renal tubular injury.

Sepsis is a serious infectious disease leading to multiple organ failure and characterized by high morbidity, rapid progression, and high mortality [16]. Annually, sepsis strikes about 50 million individuals globally, with a mortality rate exceeding 1/4 [17,18]. Among the leading causes of death in sepsis patients, acute renal failure is especially devastating [19]. Sepsis-associated acute kidney damage (SA-AKI) is a prevalent occurrence in critical patients and pertains to sepsis or septic shock affecting the kidneys, leading to a gradual deterioration in renal function [20]. SA-AKI occurs in up to 40 to 50 percent of patients and has a devastating effect on the prognosis. 18% of SA-AKI patients died in hospitals [21]. It has been considered an independent mortality risk factor in septic patients [22]. Early management to protect the kidney and lessen renal injury is critical for improving the outcomes. SA-AKI encompasses a range of intricate pathophysiological mechanisms, such as endotoxemia, renal hypoperfusion, nephrotoxic substance synthesis, inflammatory responses, oxidative stress, and others [2,23]. Among them, endotoxin, as represented by LPS, is the primary factor in the development of renal injury [24].

LPS constitutes a constituent of the external membrane of gram-negative bacterial cell walls, which is comprised of lipid and polysaccharide moieties. It is classified as an endotoxin and serves as a significant antigen, eliciting pronounced inflammatory responses [25,26]. LPS induces deleterious disorders through intricate mechanisms. During the inflammatory response generated by LPS, the activation of macrophages is initiated by intracellular signaling cascades, resulting in the creation and release of pro-inflammatory cytokines [27]. Numerous studies have demonstrated that LPS induces acute renal injury by primarily causing persistent damage to tubular epithelial cells. The excessive production of cytokines results in prolonged harm to tubular epithelial cells, ultimately causing apoptosis and necrosis of renal tubular cells [28-30]. Our study confirmed that LPS directly induced severe injury in tubular epithelial cells, characterized by tubular epithelial cell edema, necrosis, and shedding. Subsequently, the obstruction of tubular lumens occurs, leading to the deterioration of renal function and the advent of acute renal failure. The exploration of strategies to mitigate LPS-evoked tubular injury is crucial in preventing the development of SA-AKI.

Figure 7. Schematic representation of the mechanisms by which OPC mitigates LPS-induced renal tubular injury. LPS shows significant toxicity in renal tubules, which is attenuated by OPC. LPS inhibits the phosphorylation of PI3K/AKT, which causes the activation of downstream apoptosis-related signaling molecules and accelerates cell death. It also stimulates the NFκB signaling pathway, increases the production of inflammatory cytokines, and aggravates the inflammatory response. OPC reverses these alterations. Additionally, OPC mitigates LPS-induced oxidative stress injury by recovering SOD activity, CTA activity, GSH content, and inhibiting the overproduction of MDA. OPC, oligomeric proanthocyanidin; LPS, lipopolysaccharide; NFκB, nuclear factor κB; GSH, glutathione; SOD, superoxide dismutase; CTA, catalase; MDA, malondialdehyde; TNF-α, tumor necrosis factor-alpha; IL-1β, interleukin-1 beta; IL-6, interleukin-6; IL-10, interleukin-10; ROS, reactive oxygen species.

OPC is a naturally derived flavonoid defined as proanthocyanidins with small molecular weights, present in almost all edible colored plant flowers and fruits, and particularly abundant in grape seed bark and lande pine bark. It is also widely distributed in various traditional herbals like Perilla frutescens leaves, hawthorn, Lycium barbarum, and Salvia miltiorrhiza Bunge, and is valued for its nutritional and beneficial biological properties, including antioxidant, anti-inflammatory, anticancer, anti-aging, hypolipidemic, hypoglycemic, and cardiovascular and cerebrovascular protective activities [8,31]. Through its capacity to scavenge free radicals, reduce oxidative stress, and decrease inflammatory factors, OPC has been discovered to possess therapeutic potential for various diseases [8]. Our investigation shows that OPC exhibits a promising protective role in the kidney, leading to a significant reduction in LPS-induced edema, necrosis, shedding, and other detrimental alterations in renal tubules. Nevertheless, the precise mechanism remains unclear. We further determine the potential mechanism based on the RNA sequencing analysis. Through the identification of differentially expressed genes, we observed that the impact of OPC on LPS-induced injury primarily involved oxidative stress, the inflammatory response, and various signaling pathways closely associated with cell survival. Through validating the indicated signaling pathways, we discovered that OPC mitigated LPS-induced renal injury mainly through its antioxidant and anti-inflammatory effects, as well as the activation of the PI3K/AKT signaling pathway and inhibition of the NFκB signaling pathway.

Oxidative stress injury represents a significant etiological factor in the development of LPS-induced renal injury [32]. Oxidative stress is characterized by an imbalance between the production of ROS and the body's antioxidants, resulting in the accumulation of oxidative intermediates, as well as disruption of cellular defense mechanisms against oxidative damage, leading to heightened levels of intracellular peroxides, subsequently initiating a cascade of cellular impairments [33]. Oxidative stress induces peroxidation of the cell membrane, thereby compromising the membrane's structural integrity [34]. Oxidative stress induces damage to cellular organelles, such as mitochondria and endoplasmic reticulum, leading to structural and functional impairments. Additionally, it can trigger the activation of programmed cell death pathways, contributing to the development of tubular and interstitial fibrosis [35]. Our study examined the effects of LPS on the common oxidative stress markers in renal tissue, revealing a decrease in antioxidant capacity and an increase in lipid peroxidation. Nevertheless, it was observed that OPC exhibited noteworthy antioxidant properties and reinstated the antioxidant activity of the kidney, implying that the antioxidant property of OPC is pivotal in its capacity to safeguard the renal tubules.

Akt is recognized for its substantial regulatory influence on various biological processes closely linked with cell survival [36]. AKT participates in the wortmannin-sensitive pathway involving the PI3 kinase and is triggered by insulin as well as a number of growth and survival factors. By phosphorylating and inactivating a number of targets, including FoxO1, Bim, Bcl2, Bax, and others, Akt prevents cell death and encourages cell survival [37,38]. The induction of renal injury by LPS leads to apoptosis, whereas the activation of the PI3K/AKT signaling pathway can mitigate apoptosis and safeguard renal tissue from injury by suppressing the expression of apoptosis-related proteins [39]. In addition, activation of the PI3K/AKT signaling pathway has been observed to mitigate inflammatory damage in renal tissue by suppressing the synthesis of inflammatory mediators, including TNF-α and IL-6 [40]. It also enhances the migratory and proliferative capabilities of tubular epithelial cells, thereby facilitating the process of tubular repair and regeneration [41]. By recovering the activity of the AKT signaling pathway, LPS-induced renal damage can be reduced, offering a potential way to alleviate sepsis-associated renal injury linked to this pathway [42]. Our study revealed that LPS significantly inhibited PI3K/AKT phosphorylation. In contrast, OPC recovered these changes, indicating that the PI3K/AKT pathway plays a crucial role in the tubular protection afforded by OPC. OPC could promote the survival of renal tubular cells by promoting PI3K phosphorylation, thereby restoring the phosphorylation activity of AKT and inhibiting the expression of apoptosis-related molecules such as FoxO1, Bim, Bcl2, and Bax.

The exaggerated inflammatory response plays a significant role in the development of LPS-induced renal injury. NFκB serves as a crucial transcription factor in modulating the immune response to infection and is known to have a significant impact on the development of LPS-induced renal injury [43]. NF-kB is activated by various stimuli, such as inflammatory irritation, hyperglycemia, obesity, and oxidative stress [44,45]. When in an inactive state, the NFκB protein is situated within the cytoplasmic region of the cell, where it interacts with its inhibitory protein IκB to establish a complex. Nevertheless, following the stimulation of LPS, the IκB protein undergoes phosphorylation and subsequent degradation, thereby facilitating the release and translocation of NFκB into the nucleus [46]. Within the nucleus, NFκB that has been activated has the ability to attach itself to particular DNA sequences, thereby facilitating the transcription process of numerous genes associated with inflammation, including TNF-α, IL-1β, and IL-6. The secretion and subsequent release of these pro-inflammatory factors induce an excessive inflammatory response within the renal tissue, thereby exacerbating renal impairment [47]. Moreover, NFκB could initiate a cascade of apoptosis signaling pathways and regulate the overall viability of cells. The activation of NFκB induced by LPS has been observed to potentially contribute to apoptosis and necrosis in the kidney, thereby exacerbating renal function [48]. The NFκB signaling pathway is controlled by the IκB kinase (IKK) complex, which functions as a catalytic component of the IKK kinase and triggers the activation of NFκB kinase through phosphorylation modifications. Our investigation revealed that the phosphorylation of IKKα/β arose when LPS blended with ATP, which followed by NFκB phosphorylation. Interestingly, the activation of IKKα/β was considerably suppressed by OPC, leading to the inhibition of NFκB phosphorylation and the reduction of inflammatory factor release. OPC possesses a propensity for exerting a beneficial anti-inflammatory influence and safeguarding renal tubules through the mitigation of the inflammatory response via inhibiting the NFκB signaling pathway. Targeting NFκB may prove to be a promising therapeutic approach for managing renal injury caused by LPS.

In conclusion, our study revealed that OPC exhibited significant efficacy in attenuating LPS-induced renal tubular injury due to its ability to counteract oxidative stress, suppress inflammatory responses, inhibit the NFκB pathway, and activate the PI3K signaling pathway. Elucidating the precise tubular protective effect and potential underlying mechanism of OPC against LPS-induced renal injury offers more promising prospects for the clinical management of sepsis-associated acute renal injury.

Limitations

Although we found that OPC plays a positive protective role in LPS-induced renal injury through its antioxidant and anti-inflammatory activity and effects on PI3K-AKT and NFκB signaling pathways using RNA sequencing, there are many differentially gene-enriched pathways, and we only validated some signaling pathways based on our team's resources. We cannot rule out the possibility of other relevant protective mechanisms in OPC. In addition, the roles of oxidative stress, inflammation, AKT, and NFκB signaling pathways involved in LPS-induced renal injury have been confirmed in much other literature. The causal relationship still needs additional study in the future.

The authors appreciate all the participants providing supports for this study.

The authors declare no conflicts of interest.

This study was financially supported by the Jiangsu Province TCM Science and Technology Development Program Contract (QN202326 to Qijing Wu) and the Huai'an Health Research Project (HAWJ202106 to Qijing Wu, HABL202252 to Enhui Cui).