Ursodeoxycholic acid protects against sepsis-induced acute kidney injury by activating Nrf2/HO-1 and inhibiting NF-κB pathway

Abstract

Background

Ursodeoxycholic acid (UDCA), traditionally recognized for its hepatoprotective effects, has also shown potential in protecting kidney injury. This study aimed to evaluate the protective effects of UDCA against sepsis-induced acute kidney injury (AKI) and to elucidate the underlying mechanisms.

Methods

Sixty male C57BL/6 N mice were utilized to establish a sepsis-induced AKI model through intravenous injection of lipopolysaccharides (LPS, 10 mg/kg). UDCA (15, 30, and 60 mg/kg) was administered intraperitoneally once daily for 7 days before LPS injection. Kidney injury was evaluated by HE staining and biochemical markers, including serum creatinine (Cr), blood urea nitrogen (BUN), urinary protein, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), N-acetyl-β-D-glucosaminidase (NAG), and retinol binding protein (RBP). Oxidative stress parameters and nuclear factor erythroid 2-related factor 2 (Nrf2)/ heme oxygenase-1 (HO-1) pathway, pro-inflammatory cytokines and nuclear factor-kappa B (NF-κB) pathway were also evaluated. Additionally, HK-2 cells were treated with LPS in vitro, and cell viability and apoptosis were detected using CCK-8 kit and flow cytometer, respectively.

Results

UDCA significantly attenuated LPS-induced renal histopathological damage and improved renal function, as evidenced by reduction in serum Cr, BUN, and urinary protein levels. UDCA also up-regulated the protein expression of zonula occludens-1 (ZO-1) and Ezrin in the kidney, and reduced the urinary levels of NGAL, KIM-1, NAG, and RBP. Moreover, UDCA inhibited NF-κB p65 phosphorylation and reduced pro-inflammatory cytokines levels (TNF-α, IL-1β, and IL-6) in both serum and kidney. UDCA alleviated oxidative stress by activating the Nrf2/HO-1 pathway in the kidney. In vitro, UDCA reduced LPS-induced cell injury and apoptosis in HK-2 cells, with these protective effects being blocked by the Nrf2 inhibitor ML385.

Conclusions

Our present study demonstrated that UDCA exerts protective effects against sepsis-induced AKI by attenuating oxidative stress and inflammation, primarily through the activation of the Nrf2/HO-1 pathway and inhibition of the NF-κB pathway. These findings highlight the therapeutic potential of UDCA in preventing sepsis-induced AKI.

Introduction

Sepsis, which is characterized by a severe inflammatory response to infection, is the leading cause of death in critically ill patients [1, 2]. Typically, sepsis causes multi-organ failure, including acute kidney injury (AKI), and patients with AKI have an especially high mortality rate [3]. Previous reports showed that sepsis-associated AKI accounts for 45–70% of total AKI events, and about 60% of patients with sepsis developed AKI [4, 5]. Sepsis-associated AKI led to the release of inflammatory cytokines, damaged renal tubular epithelial cells, and rapid deterioration of renal function [6]. Current treatment strategies for sepsis-induced AKI include fluid resuscitation, vasopressor support, renal replacement therapy (RRT), and drugs interventions such as antibiotics, diuretics, vasodilators, and anti-inflammatory agents. However, these approaches are predominantly supportive and have notable limitations. Fluid resuscitation risks fluid overload, vasopressors may impair renal blood flow, and diuretics and vasodilators have shown inconsistent benefits in clinical trials [7–8]. Thus, there is an urgent need to develop novel effective therapeutic agents to mitigate sepsis-induced AKI.

Inflammation and oxidative stress play pivotal roles in lipopolysaccharide (LPS) induced sepsis-associated AKI [9–10]. The NF-κB pathway is a central mediator of the inflammatory response in AKI. Upon LPS stimulation, NF-κB is activated through the phosphorylation, initiating the transcription of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, leading to the inflammatory response and kidney injury [11–12]. The inhibition of NF-κB signaling has been proven to alleviate sepsis-induced AKI [9]. Concurrently, oxidative stress overwhelms the cellular antioxidant defenses and exacerbates AKI. The Nrf2/HO-1 pathway serves as a critical counter-regulatory mechanism against oxidative damage [13–14]. The activation of Nrf2 induces the expression of cytoprotective genes, including HO-1 [15]. HO-1 mitigates oxidative stress by degrading heme into biliverdin, carbon monoxide, and free iron, all of which possess antioxidant properties. In addition, HO-1 also exhibits anti-inflammation effect by inhibiting the transcription and nuclear translocation of NF-κB [16, 17]. The interplay between NF-κB-mediated inflammation and Nrf2/HO-1-driven antioxidative responses determines the severity of LPS-induced AKI. Therapeutic strategies that activate Nrf2/HO-1 pathway while simultaneously inhibiting NF-κB activation hold promise in attenuating the inflammatory and oxidative stress, potentially improving outcomes in sepsis-induced AKI.

Ursodeoxycholic acid (UDCA) is a type of hydrophilic bile acid and has been widely used as a hepatoprotective drug in clinic. Previous studies indicated that UDCA has anti-inflammation [18], anti-apoptosis [19], and anti-oxidative activities [20]. UDCA could stimulate Nrf2-mediated hepatocellular transport, detoxification, and antioxidative stress systems [21], and alleviate hepatotoxicity by activating Nrf2 signaling [22]. UDCA also alleviated inflammation by inhibiting NF-κB pathway [23]. Besides the hepatoprotective effects, the reno-protective effects of UDCA has been also verified in cisplatin-induced kidney injury [24], gentamicin-induced nephrotoxicity [25], obstructive jaundice-induced kidney injury [26], and diabetic nephropathy [27,28,29]. However, whether UDCA could protect against sepsis-induced AKI remains unclear. In the present study, a mouse model of AKI and HK2 cells treated with LPS were used to investigate the reno-protective effects of UDCA and explore its underlying mechanism based on the Nrf2/HO-1 and NF-κB pathway.

Materials and methods

Animals

Sixty male C57BL/6 N mice (7–8 weeks age) were purchased from Shanghai Slac Laboratory Animal Co., Ltd. (Shanghai, China). All mice were housed in a 12-h dark/light cycle, temperature (22 ± 2 °C) and humidity-controlled environment with unlimited access to water and food. All the procedures were performed following the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978) and approved by the Ethics Committee of the Experimental Animals of the Navy Medical University (DWLL-202303011).

Chemicals and reagents

LPS (Escherichia coli serotype 055: B5, No. L-2880), dexamethasone (DXMS, No. D4902), DMSO (No. D2650), and Penicillin-Streptomycin solution (No. P4333) were purchased from Sigma-Aldrich (St. Louis, MI, USA). Primary rabbit antibodies for NF-κB p-p65 (Ser536, No. 3033), NF-κB p65 (No. 8242), Nrf2 (No. 12721), HO-1 (No. 43966), and β-actin (No. 4970) were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit antibodies for zonula occludens-1 (ZO-1, No. 21773-1-AP) and Ezrin (No. 67627-1-Ig) were purchased from Proteintech (Wuhan, China). HRP-conjugated goat anti-rabbit secondary antibody (No. SA00001-2) was purchased from Proteintech (Wuhan, China). UDCA (No. U104241) was purchased from Aladdin (Shanghai, China). ML385 (99.96%, No. HY-100523), a selective Nrf2 inhibitor, was obtained from MCE (Shanghai, China). ANNEXIN V-FITC/PI kit (No. CA1020), DMEM/F12 medium (No. D8437), and fetal bovine serum (FBS, No. S9010) were purchased from Solarbio Science & Technology CO., LTD. (Beijing, China). QuantiNova SYBR Green (No. 208054) was purchased from Qiagen (Hilden, Germany). Cell Counting Kit-8 (CCK-8, No. CK04) was purchased from DOJINDO Laboratories (Tokyo, Japan). HK-2 cells (No. CC4008) were obtained from Guangzhou Cellcook Biotech Co., Ltd (Guangzhou, China).

Experimental design

After one week of preliminary adaptation, the mice were randomly divided into 6 groups using a computerized random number generator (10 mice per group): Control (Saline), LPS-vehicle (10 mg/kg), DXMS (5 mg/kg dexamethasone), UDCA-15 (15 mg/kg), UDCA-30 (30 mg/kg), and UDCA-60 (60 mg/kg) groups. The randomization process ensured that each mouse had an equal chance of being assigned to any treatment group, thereby minimizing bias and enhancing the validity of the results. According to the 3R principles and statistical rigor (effect size 0.4, significance level 0.05, 80% power), a sample size of 10 mice per group ensures reliable results while minimizing animal use. This approach allowed for adequate statistical efficacy to detect significant differences between treatment groups while adhering to ethical guidelines for experimental animal research. DXMS was dissolved in sterile saline and UDCA was suspended in sterile saline. Before each injection, UDCA was thoroughly sonicated and vortexed to ensure a uniform suspension. DXMS and UDCA were injected intraperitoneally once daily for 7 days, and LPS was injected intravenously trough the tail vein of mice 1 h after the last injection of UDCA, and the control group was injected with equal volume of sterile saline (Fig. 1). Based on the strong anti-inflammatory and antiendotoxin effects, DXMS was selected as a positive control drug. The dose of LPS and DXMS was selected based on previous study [30]. Previous studies indicated that 40 mg/kg of UDCA can effectively alleviate diabetic nephropathy by reducing oxidative stress [27], and 60 mg/kg of UDCA can protect against cisplatin-induced AKI in mice [24]. Therefore, 15, 30 and 60 mg/kg of UDCA was chosen in our present study. After collecting blood and 24-h urine, all mice were sacrificed by decapitation under anesthesia using tibromethanol (250 mg/kg, ip) which purchased from Sigma-Aldrich (No. T48402). The experimental treatments were conducted by specialized technical staff at the Experimental Animal Center in No. 971st Hospital of the People’s Liberation Army Navy, who were blinded to the study’s purpose, content, and treatment drugs.

Fig. 1

Timeline of the experimental schedule

Renal function

After LPS injection, all mice were transferred into a metabolic cage to collect 24-h urine. The blood was collected from retro-orbital and centrifuged at 3000 rpm for 10 min to get serum. The urine protein (No. C035-2-1), serum creatine (Cr, No. C011-1-1), and blood urea nitrogen (BUN, No. C013-2-1) were analyzed using biochemical kits (Jiancheng, Nanjing, China). Briefly, urine protein was measured though a modified acidic dye-binding method, which determines concentration based on dye absorption at 595 nm. Serum Cr was analyzed using the alkaline picrate method, in which creatinine forms a colored complex with alkaline picrate, with absorbance measured at 520 nm. BUN was detected using the diacetyl monoxime method, which producing a colored product measured at 546 nm.

Histopathological examination

Three mice were randomly selected for renal histopathological examination. Kidney tissue were harvested and fixed in 10% formaldehyde for 24 h at room temperature. Tissue sections of 5 μm thickness were cut into the slides (4–6 sections were taken from each mouse) and stained with hematoxylin and eosin (H&E). The pathological changes in the cortex of kidney tissues were observed by an optical microscope (90i, Olympus, Japan).

Oxidative stress parameters

To determine oxidative stress parameters, kidney cortex tissues (approximately 100 mg) were homogenized in 1 mL of ice-cold physiological saline and centrifuged at 3 000 g for 10 min to obtain the supernatant. A 10 µL of the supernatant was used to measure protein concentration with a bicinchoninic acid (BCA) kit (No. PC0020, Solarbio, Beijing, China), and all oxidative stress parameters were normalized based on protein concentration. The BCA assay is based on proteins can reduce Cu²⁺ to Cu⁺, forming a purple complex with bicinchoninic acid. Absorbance of this complex was measured at 562 nm using a microplate reader (Imark, Bio-Rad, Hercules, CA, USA), and protein concentration was calculated from a standard curve.

SOD activity

SOD activity was assessed using the xanthine oxidase method, where SOD inhibits the reduction of nitroblue tetrazolium (NBT). Absorbance was measured at 560 nm using a microplate reader, and SOD activity was expressed as U/mg of protein (No. A001-3-2, Jiancheng, Nanjing, China).

MDA Content

MDA levels were measured using the thiobarbituric acid (TBA) reactive substances (TBARS) method, which forms a colored complex with MDA. The absorbance of this complex was measured at 532 nm using a microplate reader and expressed as nmol/mg of protein (No. A003-1-2, Jiancheng, Nanjing, China).

GSH Content

The GSH assay is based on a reaction with 2-nitrobenzoic acid, producing a yellow product with absorbance measurable at 412 nm using a microplate reader. GSH content was calculated and expressed as µmol/mg of protein (No. A006-2-1, Jiancheng, Nanjing, China).

Enzyme-linked immunosorbent assay (ELISA)

To measure the concentrations of IL-1β (No. EK0392), IL-6 (No. EK0410), and TNF-α (No. EK0526) in kidney and serum, the ELISA method was performed using commercially available kits for cytokine detection (Boster, Wuhan, China). The levels of neutrophil gelatinase-associated lipocalin (NGAL, No. CSB-E09447m) in serum and urine, kidney injury molecule-1 (KIM-1, No. CSB-E08519m), N-acetyl-β-D-glucosaminidase (NAG, No. CSB-E09412m), and retinol binding protein (RBP, No. CSB-E11722m) levels in urine were detected using ELISA kits (CUSABIO, Wuhan, China). The preparation of all reagents and protocol were followed according to the manufacturer’s instructions. The absorbance was read using ELISA reader at 450 nm and 570 nm dual filters.

Western blot

Total proteins in kidney cortex tissues were extracted with RIPA buffer (No. R0010, Solarbio) containing phenylmethylsulfonyl fluoride (PMSF. No. P0010, Solarbio) and phosphatase inhibitor cocktail (No. P1260, Solarbio). The protein concentration was measured using the BCA method. Then, 50 µg of total proteins was separated on 10% SDS-PAGE, initially at 40 V for 30 min, followed by 80 V for 90 min. Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (No. IPVH00010, Millipore, Shanghai, China) at 200 mA for 100 min using a wet transfer method. The membranes were blocked in 5% non-fat milk at room temperature for 2 h, followed by incubation with primary antibodies against NF-κB p65 (1:1000), phosphor-NF-κB p65 (1:1000), Nrf2 (1:1000), HO-1 (1:1000), ZO-1 (1:2000), Ezrin (1:1000), and β-actin (1:5000) at 4 °C overnight. After three washes with PBST (5 min each), the membranes were incubated with an HRP-conjugated secondary antibody (1:30 000) for 1 h at room temperature. Finally, the bands were visualized using an enhanced chemiluminescence (ECL) kit (No. WBKLS0500, Merck, Darmstadt, Germany). The relative protein levels were normalized to β-actin, and Image J (v1.53, National Institutes of Health, Bethesda, MD, USA) was used to quantify relative optical density.

Cell culture

HK-2 cells were cultured in DMEM/F12 medium containing 10% FBS and 1% penicillin-streptomycin solution at 5% CO₂ and 37 °C. The seventh to eighth generation cells were used in this study. The cell injury model was established by incubating the HK-2 cells with 5 µg/mL LPS [31]. (1) To screen for compounds that provide significant protection against LPS-induced injury in HK-2 cells, we randomly selected 12 different compounds (designated CC1 to CC12) from the drug library in our laboratory. HK-2 cells were seeded in a 96-well plate at a density of 10⁴/well and cultured for 24 h. The cells were pre-treated with 20 µM of each compound for 1 h before being exposed to LPS for an additional 24 h. (2) To access the cytotoxicity of UDCA, HK-2 cells were seeded in 96-well plate at a density of 10⁴/well and cultured for 24 h. They were then treated with various concentrations of UDCA (2.5, 5, 10, 20, 40, and 80 µM) for 24 h. (3) HK-2 cells were seeded in a 96-well plate at a density of 10⁴/well and cultured for 24 h. The cells were pre-treated with DXMS (1 µM), UDCA ((10, 20, and 40 µM), or ML385 (10 µM) for 1 h before being treated with LPS for 24 h. The control group cells were treated with an equal concentration (0.1%) of DMSO as the solvent control.

Cell viability

After treatment with LPS for 24 h, the medium was replaced and 10 µL CCK-8 solution were added and cultured for 1 h. The absorbance was measured at 450 nm by microplate reader.

Cell apoptosis

For cell apoptosis assay, HK-2 cells were seeded in a 6-well plate at a density of 10⁵/well and cultured for 24 h. The cells were pre-treated with UDCA (40 µM) or ML385 (10 µM) for 1 h, followed by treatment with LPS (5 µg/mL) for an additional 24 h. After collecting the cells by centrifugation at 1000 rpm for 10 min, cells were resuspended in PBS, and 5 µL Annexin V/FITC dye was added. After incubating at room temperature for 5 min in the dark, followed by the addition of 5 µL of PI dye, the apoptosis rate was detected using a flow cytometer (CytoFLEX, Beckman, Brea, CA, USA). The parameters were set as FITC (488 nm excitation, 525 nm emission) for Annexin V and PI (488 nm excitation, 610 nm emission). Gating was performed using forward and side scatter to identify viable, early apoptotic (Annexin V⁺/PI⁻), and late apoptotic (Annexin V⁺/PI⁺) cells. Analysis was conducted with FlowJo software (v10.8, FlowJo LLC, Ashland, OR, USA).

Statistical analyses

All data are represented as mean ± S.E.M. Prior to analysis of variance, the normality and homogeneity of variances were checked using Shapiro-Wilk test. If the assumptions of ANOVA were not met, appropriate data transformations were performed using the log transformation method. One-way ANOVA followed by Turkey’s HSD post hoc test using SPSS 18.0 (IBM, Chicago, IL, USA) was conducted to analyze the statistic differences. A value of P < 0.05 was considered statistically significant.

Results

UDCA protects against LPS-induced injury in HK-2 cells

To screen for compounds that can prevent LPS-induced injury in renal tubular epithelial cells, HK-2 cells were cultured in vitro and treated with LPS. As shown in Fig. 2A, among the 12 compounds tested, CC-9 (UDCA) exhibited the best protective effect (P < 0.001). Further investigation revealed that UDCA at a concentration of 80 µM significantly reduced cell viability, indicating cytotoxicity (Fig. 2B). In contrast, UDCA at concentrations of 20 and 40 µM significantly increased cell survival under LPS exposure (P < 0.01) (Fig. 2C).

Fig. 2

The effects of UDCA on LPS-induced injury in HK-2 cells. (A) Cell viability (Treated with 12 different compounds at concentrations of 20 µM). (B) Cell viability (Treated with UDCA at concentrations of 2.5, 5, 10, 20, 40, and 80 µM, respectively). (C) Cell viability (10, 20, and 40 µM UDCA, and 5 µg/mL LPS). Data were represented as mean ± S.E.M. ^###p < 0.001 v.s. Control group; ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 v.s. LPS-vehicle group. n = 6

UDCA protects the renal dysfunction and pathological damage induced by LPS in mice

To investigate the effects of UDCA on renal function, the serum levels of Cr and BUN, and 24-h urinary protein were detected. The results showed that LPS caused a significant increase in the serum BUN (P < 0.001), Cr (P < 0.001), and 24-h urinary protein (P < 0.001) compared with control group. Treatment with UDCA and DXMS significantly prevented these changes caused by LPS (P < 0.05) (Fig. 3A-C).

To further access kidney injury, NGAL and KIM-1, two early markers of AKI, were detected using ELISA kits. The NGAL levels in serum and urine were higher in LPS group than control group (P < 0.001), while reduced by UDCA (P < 0.01) and DXMS (P < 0.001) (Fig. 3D-E). In addition, the urinary KIM-1 level was also increased by LPS (P < 0.001) but reduced by UDCA (P < 0.05) and DXMS (P < 0.01) (Fig. 3F).

The results of renal histopathological staining displayed an obvious atrophy of glomeruli and renal tubules, and infiltrated inflammatory cells in LPS group. Both UDCA and DXMS improved renal pathological changes and protected against LPS-induced renal injury (Fig. 3G).

Fig. 3

The effects of UDCA on LPS-induced kidney injury in mice. (A) Serum creatine levels. (B) Serum BUN levels. (C) Urinary proteins levels in 24 h. (D) Serum NGAL levels. (E) Urinary NGAL levels. (F) Urinary KIM-1 levels. (G) HE staining of the kidney (Upper: ×400; Lower: magnification 4 times). Data were represented as mean ± S.E.M. ^###p < 0.001 v.s. Control group; ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 v.s. LPS-vehicle group. n = 10. Bar: 50 μm

UDCA protects the renal tubular epithelial cell polarity and brush border damage induced by LPS in mice

To investigate the protective effects of UDCA on the renal tubular epithelial cell polarity and brush border integrity, the NAG activity and RBP levels in urinary were detected using ELISA kits, and the protein expression of ZO-1 (a specific marker of tubular epithelial cell polarity) and Ezrin (a specific marker of brush border integrity) were detected using Western blot. As displayed in Fig. 4, LPS significantly increased urinary NAG activity (P < 0.001) and RBP levels (P < 0.001), as well as down-regulated ZO-1 (P < 0.01) and Ezrin (P < 0.05) protein expression compared to the control group, while UDCA (P < 0.05) and DXMS (P < 0.01) significantly prevented these changes induced by LPS.

Fig. 4

The effects of UDCA on the renal tubular epithelial cell polarity and brush border integrity in LPS-induced AKI mice. (A) The urinary NAG activity. (B) The urinary RBP level. (C) The immunoblot bands of ZO-1 and Ezrin. (D) The relative expression of ZO-1 protein. (E) The relative expression of Ezrin protein. Data were represented as mean ± S.E.M. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 v.s. Control group; ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 v.s. LPS-vehicle group. n = 10 for A-B; n = 3 for C-E

UDCA decreases LPS-induced serum and renal inflammations in mice

To investigate the effects of UDCA on inflammatory response induced by LPS, the levels of IL-1β, IL-6, and TNF-α in kidney and serum were detected by ELISA kits. The results showed that LPS significantly increased these cytokines levels in kidney and serum (P < 0.01), while UDCA and DXMS significantly reduced these (P < 0.05) (Fig. 5).

Fig. 5

The effects of UDCA on the serum and renal cytokine levels in LPS-induced AKI mice. (A) Serum IL-1β levels. (B) Serum IL-6 levels. (C) Serum TNF-α levels. (D) Renal IL-1β levels. (E) Renal IL-6 levels. (F) Renal TNF-α levels. Data were represented as mean ± S.E.M. ^##p < 0.01, ^###p < 0.001 v.s. Control group; ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 v.s. LPS-vehicle group. n = 10

UDCA inhibits LPS-induced activation of NF-κB signaling in kidney

To explore the protective mechanism of UDCA on AKI induced by LPS, we detected the protein expression of NF-κB. The results were shown in Fig. 6, LPS significantly up-regulated the protein levels of phospho-NF-κB p65 in kidney (P < 0.001), both DXMS (P < 0.001) and 60 mg/kg UDCA (P < 0.01) prevented this change induced by LPS.

Fig. 6

The effects of UDCA on the renal NF-κB signaling in LPS-induced AKI mice. (A) The immunoblot bands of NF-κB. (B) The relative expression of total NF-κB protein. (C) The relative expression of p-NF-κB protein. Data were represented as mean ± S.E.M. ^###p < 0.001 v.s. Control group; ^**p < 0.01, and ^***p < 0.001 v.s. LPS-vehicle group. n = 3

UDCA reduces oxidative stress by activating Nrf2/HO-1 pathway in kidney

To explore whether UDCA inhibited NF-κB by activating Nrf2/HO-1 pathway and reducing oxidative stress, we detected the SOD activity, GSH and MDA levels, and the protein levels of Nrf2 and HO-1 in kidney. As shown in Fig. 7, LPS reduced the SOD activity and GSH levels, as well as increased MDA levels in kidney, and inhibited the protein expression of Nrf2 (P < 0.001) and HO-1 (P < 0.05). However, UDCA significantly up-regulated Nrf2 and HO-1 protein levels in kidney (P < 0.05).

Fig. 7

The effects of UDCA on the renal Nrf2/HO-1 signaling in LPS-induced AKI mice. (A) The immunoblot bands of Nrf2 and HO-1. (B) The relative expression of Nrf2 protein. (C) The relative expression of HO-1 protein. (D) The molecular docking of UDCA and Nrf2. Data were represented as mean ± S.E.M. ^#p < 0.05, ^###p < 0.001 v.s. Control group; ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 v.s. LPS-vehicle group. n = 3

Nrf2 inhibitor blocks the protective effects of UDCA on LPS-induced cell apoptosis in HK-2 cells

To investigate whether the protective effects of UDCA against LPS-induced cell injury are dependent on the activation of Nrf2/HO-1 pathway, HK-2 cells were pre-treated with the Nrf2 inhibitor (ML385) for 1 h. The results showed that the cell-protective effects of UDCA were significantly blocked by ML385 (P < 0.01) (Fig. 8C). Additionally, UDCA reduced LPS-induced cell apoptosis (P < 0.001), but this effect was also reversed by ML385 (P < 0.01) (Fig. 8D-E).

Fig. 8

The effects of Nrf2 inhibitor on the cell survival and apoptosis in HK2 cells treated with LPS. (A) Cell viability (Treated with 40 µM UDCA, 5 µg/mL LPS, and 10 µM ML385). (B) The apoptosis rate (40 µM UDCA, 5 µg/mL LPS, and 10 µM ML385). (C). Representative images of cell apoptosis detected using a flow cytometer. Data were represented as mean ± S.E.M. ^###p < 0.001 v.s. Control group; ^**p < 0.01, and ^***p < 0.001 v.s. LPS-vehicle group. n = 6 for A; n = 3 for B-C

Discussion

Previous studies have suggested that UDCA possesses anti-inflammation and anti-oxidative stress activities, and could alleviate kidney injury induced by cisplatin [24], gentamicin [25], obstructive jaundice [26], and diabetes [27,28,29]. However, whether UDCA could ameliorate sepsis-induced AKI remains unclear. In the present study, we for the first confirmed that UDCA could protect against LPS-induced AKI in mice, which evidenced by the reduced serum Cr and BUN levels, as well as the reduced urinary protein content. NGAL and KIM-1, two sensitive early markers [32, 33], were also reduced by UDCA. Moreover, UDCA also prevented renal pathological changes, as well as the impaired tubular epithelial cell polarity and brush border integrity, which evidenced by the increased expression of ZO-1 and Ezrin, and the reduced urinary NAG and RBP levels, further demonstrating that UDCA could be an effective therapeutic drug for the treatment of LPS-induced AKI.

Accumulating evidences supported that the activation of NF-κB signaling serves a crucial mediator in the development of sepsis-induced AKI [6, 34,35,36], and suggested that NF-κB pathway could be employed as a potential therapeutic target of AKI. NF-κB promotes the transcription of inflammation-related genes, and the released pro-inflammation cytokines further activate the NF-κB pathway and aggravate the inflammatory response [37]. In sepsis-induced AKI, the elevation of renal NF-κB was reported in proximal and distal tubules and in peritubular and glomerular capillaries [38]. NF-κB was also upregulated in the kidney in cisplatin-mediated AKI [35] and rhabdomyolysis-induced AKI [38]. These findings proved that NF-κB pathway plays an important role in pathophysiology of sepsis-induced AKI [34]. UDCA has been reported to produce anti-inflammation effect by inhibiting NF-κB signaling [23, 25]. In our present study, UDCA was confirmed to inhibit NF-κB activation and reduce the cytokines levels in serum and kidney, including IL-1β, IL-6 and TNF-α.

The Nrf2/HO-1 pathway is crucial for cellular defense against oxidative stress and inflammation, making it a key focus in the study of diseases including sepsis-induced AKI. Under normal conditions, Nrf2 is sequestered in the cytoplasm by its inhibitor, Kelch-like ECH-associated protein 1 (Keap1), where it undergoes ubiquitination and subsequent proteasomal degradation. Upon exposure to oxidative stress or compounds, Nrf2 dissociates from Keap1, translocates to the nucleus, and initiates the transcription of target genes, including glutathione peroxidase (GPx), SOD, and HO-1 [39]. HO-1 reduces oxidative stress by degrading heme into biliverdin, free iron, and carbon monoxide, each of which has antioxidant and cytoprotective effects. Moreover, there is already evidence indicates that HO-1 can directly inhibit the transcription of NF-κB. Previous studies indicated that UDCA can protect liver injury through Nrf2/HO-1 pathway [21, 22], however, whether the reno-protective effects of UDCA related to the activation of Nrf2/HO-1 pathway has not been studied. In our present study, LPS significantly inhibited the expression of Nrf2 and HO-1, and caused oxidative stress, but UDCA significantly upregulated their expression, increased SOD activity and GSH levels, reduced MDA levels, which further suggested that UDCA may exhibit reno-protective effects on LPS-induced AKI by activating the Nrf2/HO-1 pathway and thus inhibiting NF-κB-mediated inflammation response and reducing oxidative stress. There is also evidence to suggest that UDCA can disrupt the Keap1-Nrf2 interaction, possibly by modifying cysteine residues on Keap1, leading to conformational changes that release Nrf2 [40]. This disruption of the Keap1-Nrf2 complex is a crucial step in activating the Nrf2 pathway and upregulating HO-1 expression.

To verify these results, HK2 cells were cultured in vitro and treated with LPS. UDCA alone did not affect cell survival, but significantly increased cell survival and reduce cell apoptosis under LPS exposure. To investigate whether the protective effect of UDCA is related to the activation of Nrf2/HO-1 pathway, ML385, a specific Nrf2 inhibitor was co-incubated with UDCA in HK2 cells. The results showed that ML385 significantly blocked the protective effect of UDCA, and increased cell apoptosis and decreased cell survivals compared with UDCA alone group. These findings further verified that the renal protective effect of UDCA was dependent on the up-regulation of Nrf2/HO-1 pathway.

While UDCA shows promise in protecting against LPS-induced AKI via the Nrf2/HO-1 pathway, translating these findings into clinical practice presents challenges. The first challenge is determining the optimal timing for UDCA administration in sepsis. Late administration may be less effective due to irreversible damage, while early administration could hinder essential immune responses against infection. Identifying the optimal window for the protective effects of UDCA is crucial. Secondly, whether UDCA will interact with standard sepsis treatments, such as antibiotics, vasopressors, and glucocorticoids, and affect its efficacy. Finally, patient variability in response to UDCA, influenced by genetic differences, comorbidities, and disease severity, further complicates its clinical application, necessitating a personalized treatment approach. Future studies need to address these critical issues, and these issues are essential to ensure that UDCA is safely and effectively incorporated into the treatment regimen for sepsis patients.

For the future clinical trials, our findings offer valuable insights that can guide the design of future clinical trials involving UDCA in the treatment of sepsis-induced AKI. Firstly, future trials should target those patients with liver injury, or with oxidative stress and inflammation using biomarkers for selection. Stratification by inflammatory status, using markers like TNF-α or IL-6, may help identify those who could benefit most from UDCA. Outcome measures should include traditional renal function markers such as Cr and BUN, alongside sensitive biomarkers like NGAL, KIM-1, and RBP. Evaluating oxidative stress and inflammatory markers (MDA, SOD, IL-6, and TNF-α) is also crucial.

Previous pharmacokinetic studies have shown that UDCA is primarily excreted through bile secretion, exhibiting significant enterohepatic circulation with a half-life of approximately 3.5 to 5.8 days [41]. Since it is not renally excreted, its elimination is unaffected in AKI patients; however, absorption and distribution may still be altered, necessitating further detailed pharmacokinetic studies. In terms of safety, UDCA is generally well-tolerated, with mild gastrointestinal symptoms being the most common side effects in the treatment of liver diseases [42]. Nonetheless, these potential side effects should not be overlooked when considering UDCA for the treatment of AKI.

Conclusion

Our present study for the first time demonstrated that UDCA can effectively protect against LPS-induced AKI by activating Nrf2/HO-1 and inhibiting NF-κB pathway, thus reducing oxidative stress and inflammation, and improved renal function (Fig. 9). These findings revealed a new mechanism by which UDCA improve AKI and supplied a potential therapeutic target for AKI. However, this study has several limitations, including the absence of in vivo interference experiments to validate the proposed mechanism, insufficient exploration of the direct molecular targets of UDCA, and whether UDCA may act through other pathways. To address these issues, future research will utilize shRNA gene silencing, multi-omics technologies, and structural pharmacology methods such as molecular docking, surface plasmon resonance (SPR), target fishing, and molecular dynamics simulations. These approaches will provide a more comprehensive understanding of the molecular mechanisms of UDCA and its therapeutic potential.

Fig. 9

The underlying mechanism of UDCA protects against LPS-induced AKI