Introduction

Obstructive jaundice (OJ) is a common hepatobiliary surgical condition characterized by the mechanical blockage of intrahepatic or extrahepatic bile ducts due to various factors including tumors, stones, and inflammation. Patients with OJ face a significantly increased risk of perioperative complications, such as infection, sepsis, and multiple organ dysfunction syndrome, all of which can severely impact prognosis. Kidney injury (KI) is a particularly serious complication, affecting approximately 8–10% of patients with OJ during the perioperative period and carrying a mortality rate as high as 80%1. Researchers have referred to the renal damage caused by obstructive jaundice as obstructive jaundice kidney injury (OJKI). Despite its high mortality rate, no specific treatments currently exist for OJKI, and therapeutic approaches remain purely symptomatic. This lack of understanding regarding the underlying mechanisms and treatment options for OJKI presents a significant challenge in reducing patient mortality.

The G protein-coupled bile acid receptor 1 (Gpbar1) is a member of the G protein-coupled receptor family, functions as a membrane receptor for bile acids. Gpbar1 is distributed in various tissues and is activated by bile acids such as lithocholic acid, chenodeoxycholic acid, deoxycholic acid (DCA), and cholic acid. Studies have suggested that decreased Gpbar1 expression is associated with mucosal damage in the intestine and reduced proliferation of epithelial cells during OJ2. Additionally, Gpbar1 has been linked to mitochondrial-mediated inflammatory responses and the development of renal fibrosis3. However, the role of Gpbar1 in renal injury during OJ remains unclear.

NAD-dependent deacetylase sirtuin1 (SIRT1) plays a crucial role in regulating nuclear-encoded mitochondrial gene transcription, and its involvement in mitochondrial biogenesis and renal metabolism regulation is well established4. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a key transcriptional coactivator in mitochondrial biogenesis, is essential for regulating gene expression related to this process5. Tran M et al. found that PGC-1α alleviates acute kidney injury induced by ischemia-reperfusion by improving mitochondrial dysfunction via p38 signaling6. Simultaneously, studies have confirmed that the SIRT1 agonist SRT1720 can accelerate the recovery of organ function within 24 hours through SIRT1-PGC-1α-mediated mitochondrial biogenesis in renal tubular cells following acute kidney injury7. Similarly, resveratrol activation of the SIRT1-PGC-1α signaling pathway has been shown to alleviate progressive renal injury and apoptosis in diabetic nephropathy mice by enhancing mitochondrial biogenesis8,9,10. However, the role of SIRT1-PGC-1α-regulated mitochondrial function changes in the repair of OJKI remains to be explored.

In this study, we established the OJKI model by surgical ligation of the common bile duct in rats and induced the renal injury model by treating human renal tubular epithelial cells with DCA. Our findings reveal a critical role for Gpbar1 in protecting against bile stasis-induced renal injury in both in vivo and in vitro settings. Gpbar1 regulation of mitochondrial function alterations through SIRT1-PGC-1α is a potential key mechanism of renal injury in obstructive jaundice and provides a promising strategy for the prevention and treatment of OJKI.

Materials and methods

Establishment of rats OJKI model

The rats used in this study were purchased from Liaoning Changsheng Biological Co., Ltd (Changsheng Biological, China). The model was established according to the methods of Hatipoglu et al. and Kong et al.11,12. After a 7-day period of adaptive feeding in a controlled environment, 20 male Sprague-Dawley (SD) rats were randomly assigned to either the control group (n = 10) or the bile duct ligation group (n = 10). One day prior to surgery, abdominal hair was removed using a depilatory cream. Anesthesia was induced with isoflurane, and the depth of anesthesia was assessed by the absence of corneal reflex. The abdominal skin was disinfected with iodophor, and a midline incision was made to open the abdominal cavity. The intestines were gently retracted using a sterile cotton swab to expose the portal region, where the common bile duct (CBD) was identified. The CBD was double-ligated with 5 − 0 silk suture, and its distal end was single-ligated before being transected at the midpoint. In the sham-operated group, the CBD was exposed but not ligated, and the abdominal cavity was closed layer by layer using 3 − 0 silk suture. Postoperative monitoring was conducted daily. Fourteen days after surgery, the abdominal cavity was reopened along the original incision. The abdominal organs and the abdominal aorta were sequentially exposed, and the adipose tissue anterior to the spine was carefully retracted to reveal the abdominal aorta and the inferior vena cava. A total of 5 mL of blood was collected from the abdominal aorta using a blood collection needle and a vacuum blood collection tube. Immediately after blood collection, kidney and liver tissues were excised, rinsed in physiological saline, and either snap-frozen in liquid nitrogen or fixed in 4% paraformaldehyde for subsequent analysis. All animals were euthanized by cervical dislocation at the end of the procedure. We confirm that the study is reported in accordance with ARRIVE guidelines.

Biochemical, antioxidants, and ATP content detection in OJKI rats’ serum and renal tissue

Detection of liver and kidney function indicators: After standing for 2 hours, rat blood was centrifuged (3000 rpm, 10 minutes) to collect serum, and the serum was analyzed for ALT, AST, TBIL, DBIL, BUN, and CRE using an automated biochemical analyzer. Detection of antioxidant and ATP content in renal tissue: After homogenizing kidney tissue, the levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), and ATP in the kidney tissue were measured according to the instructions of the assay kit (Nanjing Jiancheng).

Immunohistochemical staining

After excising the liver and kidney tissues from the rats, the samples were immediately fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 4 μm. The paraffin sections were placed in a 60 °C oven for 2 hours, followed by deparaffinization with xylene (twice, 5 minutes each). The sections were then rehydrated through a graded ethanol series (absolute ethanol I, absolute ethanol II, 95% ethanol, 85% ethanol, and 70% ethanol, each for 5 minutes) and finally rinsed in distilled water. Antigen retrieval was performed using EDTA buffer (pH 8.0) in a microwave oven. To block endogenous peroxidase activity, sections were incubated with 3% hydrogen peroxide, followed by blocking with 5% BSA to reduce nonspecific binding. The primary antibody, appropriately diluted, was added, and the sections were incubated overnight at 4 °C. After 12 hours, the sections were incubated with an HRP-conjugated goat anti-mouse/rabbit IgG polymer secondary antibody, followed by DAB chromogen staining, and observed under a microscope. The nuclei were counterstained with hematoxylin, differentiated with 1% acid ethanol, and rinsed with water to restore the blue color. Sections were then dehydrated in graded ethanol solutions (70%, 85%, 95%, absolute ethanol I, absolute ethanol II, each for 1 minute), cleared in xylene (twice, 1 minute each), and mounted with neutral resin for microscopic imaging.

IHC scoring

IHC scoring was independently performed by two pathologists using the Immunoreactivity Score (IRS) system, which integrates staining intensity and the proportion of positively stained cells. Staining intensity was classified into four levels: 0 (negative, no staining), 1 (weak positive, light yellow), 2 (positive, brown-yellow), and 3 (strong positive, dark brown). The percentage of positive cells was categorized into four ranges: 1 point (< 10% positive cells), 2 points (11–50%), 3 points (51–80%), and 4 points (> 80%). The final IRS score was calculated as IRS = staining intensity score × percentage of positive cells.

HE staining

HE staining was performed according to the instructions provided in the HE staining kit (Solarbio, China). The procedures from section baking to gradient ethanol dehydration were the same as those used for IHC staining. The sections were then stained with hematoxylin for 10 min, followed by washing with tap water and differentiation with 1% acid ethanol. After rinsing with distilled water for 10 minutes to restore the blue color, the sections were counterstained with 0.5% eosin aqueous solution for 2 minutes. The subsequent dehydration, clearing, mounting, and imaging steps were identical to those used for IHC staining.

HE scoring

We assessed renal tissue injury scores in hematoxylin and eosin (HE)-stained kidney sections following the method described by Yang et al.13. Renal injury was classified into six levels based on the severity of tubular epithelial cell damage, detachment, loss of the brush border, and peritubular inflammatory cell infiltration. Each section was evaluated in five randomly selected fields, with each field scored from 0 to 5 (0: normal; 1: mild injury, involvement of 0–10%; 2: moderate injury, involvement of 11–25%; 3: severe injury, involvement of 26–49%; 4: very severe injury, involvement of 50–75%; 5: extensive injury, involvement of > 75%). For liver injury assessment, we referred to the Suzuki scoring system with modifications14,15,16. The injury score was based on four histological parameters with grading on a 0–4 scale (0: no abnormality; 1: mild; 2: moderate; 3: severe; 4: extremely severe). The scoring criteria considered the extent of sinusoidal expansion, disruption of lobular architecture, degree of hepatocyte swelling, and intensity of inflammatory cell infiltration, with higher scores indicating more severe injury. All evaluations were performed by two blinded investigators to ensure objectivity and consistency.

TUNEL staining

Renal tissue apoptosis was assessed using a one-step TUNEL apoptosis detection kit (Beyotime, China). Briefly, paraffin sections were deparaffinized, rehydrated, and then treated with proteinase K to permeabilize cells and expose DNA, following the manufacturer’s instructions. After PBS washing, TdT enzyme, fluorescent labeling solution, and TUNEL detection solution were added in specific proportions. Following a second PBS wash, sections were mounted using DAPI-containing mounting medium. Apoptotic cells exhibiting green fluorescence were observed under a fluorescence microscope, and five random fields were selected in each section for imaging.

Cell culture, virus transfection and establishment of DCA-injured cell model

Human renal tubular epithelial cells (HK-2) were purchased from Sanyuan Biotech (Wuhan, China) and cultured in minimum essential medium (MEM, Pricella, China) containing 10% FBS and maintained at 37 °C in an incubator with 5% CO2. Lentiviral vectors were obtained from GenePharma (Suzhou, China). HK-2 cells were cultured in 6-well plates for 24 hours and then transfected with Gpbar1 overexpression lentiviral vector or Gpbar1 knockdown lentiviral vector. The transfection system was supplemented with 5 µg/mL Polybrene (Solarbio, China) and the medium was replaced with fresh medium after 24 hours of culture. Infected cells were selected with puromycin (Beyotime, China). To simulate SIRT1 activation, inhibition, and PGC-1α inhibition, HK-2 cells were treated with 1 µM SRT1460, 1 µM EX527, or 1.5 µM SR18292 for 24 hours, respectively. The experimental group was treated with 0.3 mM DCA for 24 hours to induce cell damage and establish a DCA-injured cell model.

Analysis of cell viability

HK-2 cells (12,000 cells per well) were seeded in 96-well plates. A concentration gradient of DCA was set and the cells were stimulated with different concentrations of DCA for 24, 48, and 72 hours. After the treatment, 10 µL of CCK-8 solution was added to each well. After incubation for 2 hours at 37 °C, the absorbance at 450 nm was measured with an microplate reader.

Transmission electron microscopy ultrastructure observation

Fresh tissue was initially fixed with electron microscope fixative (Servicebio, China), then transferred to a 1% osmium tetroxide solution for secondary fixation. The samples were rinsed with phosphate buffer and dehydrated through a graded alcohol series. Acetone mixed with 812 embedding resin was used for infiltration and embedding. The resin-embedded blocks were sectioned at 1.5 μm thickness, stained with toluidine blue, and observed under a microscope for positioning. Next, ultrathin sections (60–80 nm) were cut from the resin block and stained with 2% uranyl acetate followed by 2.6% lead citrate. After drying, the sections were examined with a transmission electron microscope (JEM1400PLUS, Japan) to observe mitochondrial structure and morphology.

Measurement of cellular ROS

The transfected HK-2 cells were seeded in 6-well plates, and then DCA was used to establish a cell injury model. The intracellular ROS was measured using a reactive oxygen species detection kit (Solarbio, China). According to the manufacturer’s instructions, the fluorescent probe DCFH-DA was diluted with serum-free MEM medium, and the probe working concentration was set to 5 µM. The cells were washed twice with PBS, and 1 mL of the diluted DCFH-DA was added and placed in a 37 °C incubator for 30 minutes. The cells were washed three times with serum-free medium, and the fluorescence images were taken with a fluorescence microscope.

Mitochondrial membrane potential detection

Mitochondrial membrane potential was assessed using the JC-1 Mitochondrial Membrane Potential Detection Kit (Beyotime, China). Transfected HK-2 cells were seeded in 6-well plates for DCA injury experiments. Following the manufacturer’s instructions, the culture medium was removed, and the cells were washed twice with PBS. Then, 1 mL of complete culture medium and 1 mL of JC-1 staining working solution were added, mixed gently, and incubated at 37 °C for 20 minutes. The 5×JC-1 staining buffer was diluted to 1× with distilled water. After incubation, the supernatant was removed, and cells were washed twice with the diluted JC-1 staining buffer. Finally, 2 mL of complete culture medium was added, and the cells were examined and photographed under a fluorescence microscope.

Detection of intracellular cAMP and extracellular TFAM

HK-2 cells and transfected cells were cultured in 6-well plates and treated with DCA. After exposure, cells and supernatants were collected. For cell samples, cells were collected, resuspended in PBS, and lysed using an ultrasonic disruptor. The lysates were centrifuged to collect the supernatant, which was then diluted 1000-fold. For cell culture supernatants, the samples were centrifuged at 1000 ×g for 20 minutes to remove impurities and cell debris. The cAMP levels in cell lysates were measured using a cAMP ELISA kit (Elabscience, USA), and TFAM levels in cell supernatants were measured using a TFAM ELISA kit (ELK Biotechnology, China). Following the manufacturer’s protocol, standards and samples were added to the ELISA plate, followed by antibody working solution, and incubated at 37 °C for 45 minutes. After incubation, the antibody solution was discarded, and the plate was washed three times with wash solution. Enzyme conjugate working solution was added and incubated at 37 °C for 30 minutes. Following three additional washes, substrate solution was added and incubated at 37 °C for 15 minutes. Finally, stop solution was added, and the absorbance was measured at 450 nm using an microplate reader.

Western blot analysis

Total protein was extracted from renal tissues and HK-2 cells using a total protein extraction kit (Solarbio, China). Proteins were separated by SDS-PAGE and transferred onto PVDF membranes. To block non-specific binding, the membranes was incubated with 5% skim milk for 2 hours, followed by overnight incubation with primary antibodies and then with secondary antibodies for 1 hour. Protein bands were visualized using an enhanced chemiluminescence reagent. The primary antibodies included β-actin (Abclonal, China), Gpbar1 (Abcam, USA), SIRT1 (Abcam, USA), and PGC-1α (Abclonal, China), with HRP-conjugated goat anti-rabbit IgG secondary antibodies (Abcam, USA). Quantitative analysis of protein expression was conducted using ImageJ software, with protein levels normalized to β-actin.

Real-time quantitative PCR

Total RNA from tissues/cells was extracted using the SteadyPure Universal RNA Extraction Kit (Accurate Biology, China). cDNA was synthesized using the Reverse Transcription Premix Kit (Accurate Biology, China). qPCR was performed using the SYBR Green Pro Taq HS Premix qPCR Kit (Accurate Biology, China). All reactions were performed in triplicate, and β-actin was used as an endogenous control. The relative expression of genes was calculated using the 2 − ΔΔCT method. The primers used for PCR are listed in Supplementary Table 1.

Ethics statement

This study was performed in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines. The rat experiments and the establishment of the rat bile duct ligation model were performed using protocols approved by the Animal Ethics Committee of Dalian Medical University (approval number: AEE24044). All animal experimental procedures fully complied with the internationally recognized principles for the care and use of laboratory animals.

Statistical analysis

Statistical analysis was performed using Prism 9.5 software (GraphPad, San Diego, USA), and the data were expressed as mean ± standard deviation. One-way ANOVA analysis of variance was used for comparisons among multiple groups. Independent sample t-tests were used to compare differences between two groups. P < 0.05 was defined as reaching statistical significance.

Results

Successful establishment of rat OJKI model

To develop the OJKI model, rats were randomly divided into two groups: the control (CON) group and the bile duct ligation (BDL) group. After a 7-day period of acclimation, rats in the BDL group underwent bile duct ligation surgery, while those in the CON group had their bile ducts exposed but not ligated. Post-surgery, both groups were fed a standard diet, and after 14 days, the rats were euthanized for further analysis (Fig. 1A).

Fig. 1
figure 1

Establishment of obstructive jaundice-induced kidney injury (OJKI) rat model. (A) Schematic diagram illustrating the bile duct ligation (BDL) procedure for establishing the OJKI model in rats. (B) Histopathological staining and scoring analysis of liver and kidney tissues in control (CON) and BDL groups (Black arrows: dilated hepatic sinusoids; White arrows: swollen hepatocytes; Red arrows: inflammatory cell infiltration in liver and kidney; Green arrows: tubular damage in kidney; Blue arrows: damaged glomeruli.) (***p < 0.001). (C) Serum liver and renal function indicators (ALT, AST, TBIL, DBIL, BUN, CRE) in CON and BDL groups (*p < 0.05, **p < 0.01, ***p < 0.001). CON, control. BDL, bile duct ligation. OJKI, obstructive jaundice induced kidney injury.

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Liver and kidney tissues were collected for histological examination. Using HE staining, we observed significant structural changes in the livers of the BDL group compared to the CON group. The liver lobules in the BDL group appeared disorganized, with indistinct hepatocyte boundaries and dilated hepatic sinusoids, indicating hepatocellular swelling and tissue stress. There was also notable infiltration of inflammatory cells, leading to a marked increase in liver pathology scores in the BDL group. Similarly, HE staining of kidney tissues revealed severe disruption of the renal structure, characterized by indistinct tubular boundaries and increased infiltration of inflammatory cells. This disruption resembles the erosion of a building’s foundation, resulted in a loss of normal renal architecture. Some glomeruli exhibited swelling or structural disorganization, accompanied by capillary dilation or collapse. In certain areas, an expansion of the Bowman’s space is observed, suggesting possible impairment of filtration function. Consequently, the BDL group exhibited a significantly higher kidney pathology score compared to the CON group. (Fig. 1B). Furthermore, liver and kidney function indicators, including ALT, AST, TBIL, DBIL, BUN, and CRE, were markedly elevated in the BDL group (Fig. 1C). Bile duct ligation obstructs bile excretion, leading to the accumulation of toxic substances such as bile acids and bilirubin in the liver. This results in hepatocyte injury, characterized by cellular swelling, necrosis, sinusoidal dilation, and induction of inflammatory responses. Hepatocyte structural disorganization and elevated transaminase levels serve as direct evidence of liver injury caused by obstructive jaundice. Structural alterations in the renal tubules represent a direct manifestation of bile-induced renal injury, while glomerular changes may be secondary damage caused by tubuloglomerular feedback. These results collectively indicate substantial structural and functional damage in the liver and kidney tissues of rats, confirming the successful establishment of the OJKI model.

Mitochondrial oxidative stress and cell apoptosis are involved in OJKI

Mitochondrial dysfunction is commonly observed in acute kidney injury (AKI) and is closely associated with impaired energy metabolism, increased reactive oxygen species (ROS) production, oxidative stress, and mitophagy1,17,18,19. To assess whether renal damage caused by obstructive jaundice (OJ) is related to mitochondrial homeostasis, we conducted a series of analyses using renal tissues. First, we investigated apoptosis in renal tissues using the TUNEL assay (Figs. 2A-B). The BDL group showed a significant increase in the apoptotic index compared to the CON group, confirming that cell apoptosis occurs during OJKI. Then, we examined mitochondrial structure and morphology changes using transmission electron microscopy (Fig. 2C). In the CON group, mitochondria exhibited a normal morphology with intact cristae and no signs of swelling. In contrast, mitochondria in the BDL group displayed significant swelling and an increased size compared to those in the CON group. Moreover, their morphology was highly heterogeneous and abnormal. The mitochondrial matrix appeared more transparent, indicating reduced electron density, and the cristae lumen was markedly expanded. These ultrastructural changes suggest excessive water retention, a hallmark of mitochondrial injury. Additionally, the cristae of many mitochondria appeared indistinct, with some nearly devoid of a normal cristae structure. This may be attributed to the fusion of cristae membranes into irregular large membrane structures, resulting in the loss of typical lamellar cristae morphology. Collectively, these morphological alterations indicate severe mitochondrial damage in the renal tissues of OJ rats. To further determine whether mitochondrial oxidative stress contributes to this damage, we measured key antioxidant markers (SOD, GSH, MDA, Fig. 2D) and ATP levels in renal tissues (Figs. 2E). The BDL group demonstrated a significant reduction in SOD and GSH levels, along with a marked increase in MDA levels, indicating elevated oxidative stress. Additionally, ATP content in the BDL group was significantly lower than in the CON group, reflecting impaired energy metabolism. Collectively, our findings suggest that mitochondrial oxidative stress may play a critical role in disrupting mitochondrial homeostasis, promoting apoptosis, and contributing to renal injury during OJKI.

Fig. 2
figure 2

Obstructive jaundice induces cell apoptosis and mitochondrial damage in renal tissue. (A) TUNEL staining comparing cell apoptosis in control (CON) and bile duct ligation (BDL) rat renal tissues. (B) Quantitative analysis of cell apoptosis rate after fluorescence detection across experimental groups (***p < 0.001). (C) Transmission electron microscopy of mitochondrial morphology in rat renal tissues (Blue arrows: swollen mitochondria; Green arrows: expanded intercristal spaces; Red arrows: disrupted cristae structures). (D) Measurement of antioxidant indicators (SOD, GSH, MDA) in renal tissues (**p < 0.01, ***p < 0.001). (E) Measurement of ATP content in renal tissues (**p < 0.01). CON, control. BDL, bile duct ligation.

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Gpbar1, SIRT1, and PGC-1α expressions are downregulated during OJKI

Gpbar1 is an important cell membrane-bound bile acid receptor. It has been shown to play an important regulatory role in inflammation within multiple organs. Many studies have reported that Gpbar1 plays a protective role in alcoholic liver injury, fat-related liver injury, and drug-induced liver injury20,21,22,23,24,25. During obstructive jaundice, low expression of Gpbar1 is associated with intestinal mucosal damage and decreased epithelial cell proliferation2. The SIRT1-PGC-1α signaling pathway is an important regulatory pathway for mitochondria to resist oxidative stress, which plays a protective role in renal injury6,7. Therefore, we explored the changes in Gpbar1 and SIRT1-PGC-1α signaling pathways using immunohistochemistry, Western blotting, and qRT-PCR.

Gpbar1 is primarily expressed in the cell membrane of renal tubular epithelial cells, with strong staining observed in the brush border of proximal tubules, while a small amount is also distributed in the cytoplasm and nucleus. SIRT1 is mainly expressed in renal tubular epithelial cells (especially in proximal tubules) and is localized in both the cytoplasm and partially in the nucleus. PGC-1α exhibits positive cytoplasmic staining in the cytoplasm of renal tubular epithelial cells (Fig. 3A). There was a small amount of Gpbar1, SIRT1 and PGC-1α expression in the kidney tissue of rats in the BDL group, and their expression levels were significantly lower than CON group. Western blot and qRT-PCR results also confirmed that the BDL group showed lower levels of Gpbar1, SIRT1 and PGC-1α at the protein and mRNA levels (Figs. 3B-D). These results suggest that OJKI may be associated with the loss of the protective effect of Gpbar1, increased oxidative stress, and loss of mitochondrial homeostasis.

Fig. 3
figure 3

Obstructive jaundice decreases the expression of Gpbar1, SIRT1, and PGC-1α in rat renal tissues. (A) Immunohistochemical observation comparing the expression of Gpbar1, SIRT1, and PGC-1α between control (CON) and bile duct ligation (BDL) rat renal tissues. (B-C) Western blot analysis to compare the protein levels of Gpbar1, SIRT1, and PGC-1α between the BDL group and the CON group (*p < 0.05, ** p < 0.01). (D) The mRNA expression levels of Gpbar1, SIRT1, and PGC-1α in rat kidney tissues of BDL group compared to CON group (**p < 0.01, ***p < 0.001). CON, control. BDL, bile duct ligation.

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Gpbar1 was a key regulator in DCA-induced cell injury model

Firstly, we set the concentration gradient of DCA from 0 to 1mM and treated cells for 24, 48, and 72 hours. We calculated the IC50 of DCA at different times. The IC50 at 24 hours was 0.3 mM, so we selected the drug concentration of 0.3 mM for 24 hours to establish a DCA-induced cell injury model (Fig. 4A). Subsequently, we utilized lentivirus to establish stable cell lines with Gpbar1 overexpression or knockdown to investigate the specific role of Gpbar1 in DCA injury model (Fig. 4B). The experimental groups were divided into four subgroups: CON group, DCA group, DCA + Gpbar1 overexpression group (DCA + OV), and DCA + Gpbar1 knockdown group (DCA + KD). Next, to determine whether Gpbar1 plays a protective role in the DCA injury model, we conducted a cell viability assay. The results demonstrated a significant decrease in cell viability in the DCA group compared to the CON group. Moreover, the DCA + KD group exhibited a more pronounced decrease in cell viability compared to the DCA group. Conversely, the DCA + OV group showed an increase in cell viability compared to the DCA group (Fig. 4C). Given that Gpbar1 is a G protein-coupled receptor and cAMP is its second messenger, we assessed Gpbar1 activity in the DCA model by measuring intracellular cAMP levels. The results indicated a significant reduction in intracellular cAMP levels in the DCA group compared to the CON group. Furthermore, the DCA + KD group displayed a more notable decrease in intracellular cAMP levels than the DCA group. In contrast, the DCA + OV group exhibited an increase in intracellular cAMP levels compared to the DCA group (Fig. 4D).

Fig. 4
figure 4

Gpbar1 is the key regulator in deoxycholic acid-induced renal cell injury model. (A) The cell viability of HK-2 cells treated with different concentrations of deoxycholic acid (DCA) for 24 hours, 48 hours, and 72 hours. (B) Construction of stably transfected gpbar1 overexpression and knockdown lentivirus, and transfection into HK-2 cells to establish Gpbar1 overexpression and knockdown stable cell lines. (C) The cell viability of HK-2 cells in each group after treatment with DCA (***p < 0.001). (D) The intracellular cAMP levels of each group after treatment with DCA (***p < 0.001). (E-F) Western blot analysis with β-actin as an internal reference to compare the protein expression of Gpbar1, SIRT1, and PGC-1α in HK-2 cells across each group (*p < 0.05, **p < 0.01, ***p < 0.001). (G) qRT-PCR analysis with β-actin as an internal control to compare the mRNA expression levels of Gpbar1, SIRT1, and PGC-1α in HK-2 cells across each group (*p < 0.05, ***p < 0.001).

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As mentioned above, oxidative stress is a key factor in renal cell apoptosis during OJKI, Gpbar1 plays a protective role in bile-induced organ damage, and SIRT1-PGC-1α signaling pathway plays a protective role in oxidative stress damage to the kidney. To further explore the link between Gpbar1 and the SIRT1-PGC-1α signaling pathway, we conducted Western blotting and qRT-PCR analysis to assess the expression of these proteins in the treated cell groups. Western blot results revealed decreased Gpbar1, SIRT1, and PGC-1α protein expression in the DCA group compared to the CON group. The DCA + KD group displayed an even more pronounced decrease, while the DCA + OV group showed higher expression of these proteins compared to the DCA group (Figs. 4E-F). The results of the qRT-PCR results were consistent with the Western blot results (Fig. 4G).

In summary, our experiments provide preliminary evidence that Gpbar1 expression plays a protective role in OJKI. Furthermore, we observed significant correlated changes in the SIRT1-PGC-1α signaling pathway during the process of Gpbar1 action in OJKI.

Gpbar1 mitigates cell injury by reducing oxidative stress during obstructive jaundice

In addition, we explored whether the Gpbar1-mediated SIRT1-PGC-1α signaling pathway alleviates renal damage during obstructive jaundice by resisting oxidative stress in vitro. First, we observed the mitochondrial morphology of cells from each group using transmission electron microscopy after establishing our model. Mitochondria in the CON group exhibited a normal and well-organized structure, while those in the DCA group showed significant swelling and reduced cristae density. Moreover, the DCA + OV group displayed milder mitochondrial damage compared to the DCA group with decreased swelling and relatively regular cristae arrangement, whereas the DCA + KD group exhibited aggravated damage characterized by pronounced mitochondrial swelling, blurred and disorganized cristae, and partial cristae disappearance or vacuolization (Fig. 5A). The degree of mitochondrial damage in DCA-treated HK-2 cells appeared to change in parallel with the elevation and reduction of Gpbar1, suggesting that the Gpbar1-mediated SIRT1-PGC-1α signaling pathway may resist renal injury during obstructive jaundice by maintaining mitochondrial homeostasis. Second, to investigate the relationship between mitochondrial damage and oxidative stress, we measured intracellular ROS levels. The results showed a significant increase in average fluorescence intensity in the DCA group compared to the CON group. Moreover, the DCA + KD group exhibited a more pronounced increase in average fluorescence intensity compared to the DCA group, while the DCA + OV group showed a decrease compared to the DCA group (Fig. 5B-C). Additionally, we performed mitochondrial membrane potential experiments to assess mitochondrial function in the OJKI model. The JC-1 Ratio in the DCA group significantly decreased, indicating substantial mitochondrial damage during OJKI. Furthermore, the JC-1 ratio markedly increased or decreased in accordance with the elevation or reduction of Gpbar1 (Fig. 5D). Finally, we examined the crucial mitochondrial regulatory protein TFAM. ELISA analysis revealed that TFAM levels in the cell supernatant were higher in the DCA group than in the CON group. Moreover, the TFAM content in the DCA + KD group was elevated compared to the DCA group, and the extracellular TFAM level decreased in the DCA + OV group compared to the DCA group (Fig. 5E). These data indicate that in the DCA-induced cell injury model, Gpbar1 reduces the release of mitochondrial reactive oxygen species, thereby alleviating mitochondrial damage, reducing cell damage and inhibiting cell apoptosis.

Fig. 5
figure 5

Impact of Gpbar1 expression on cellular mitochondrial function and oxidative stress levels in the cell model induced by deoxycholic acid. (A) Transmission electron microscopy observation of mitochondrial ultrastructure in each group of cells (Blue arrows: swollen mitochondria; Red arrows: disrupted cristae structures) (scale bar 0.5 μm). (B) Fluorescence measurement of ROS levels in each group of cells. (C) Quantitative analysis of average ROS fluorescence levels in each group (**p < 0.01, ***p < 0.001). (D) JC-1 assay to detect mitochondrial membrane potential in each group of cells. (E) ELISA measurement of TFAM levels in the cell supernatant of each group (*p < 0.05, ***p < 0.001).

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Further clarification of the upstream and downstream regulatory relationship between Gpbar1, SIRT1, and PGC-1α

Next, to verify whether Gpbar1 expression can mediate the SIRT1-PGC-1α signaling pathway, we conducted a simple validation of the downstream regulation of the SIRT1 and PGC-1α pathway. HK-2 cells were treated with the SIRT1 inhibitor EX527 and the PGC-1α inhibitor SR18292, and the expression of SIRT1 and PGC-1α was analyzed by Western blot and qRT-PCR. The results showed that after treatment with EX527, the expression of both SIRT1 and PGC-1α decreased in HK-2 cells. However, after treatment with SR18292, only the expression of PGC-1α decreased while SIRT1 expression remained unaffected (Fig. 6A-D). Here, we confirmed that SIRT1 can regulate PGC-1α, and SIRT1 acts as an upstream regulatory factor for PGC-1α. Subsequently, we further explored the relationship between Gpbar1 and the SIRT1-PGC-1α signaling pathway by using HK-2 cells with stable overexpression or knockdown of Gpbar1 and treatment with the SIRT1 activator SRT1460. Results from Western blot and qRT-PCR analyses revealed that upon stimulation with a low dose of DCA, overexpression and knockdown of Gpbar1 corresponded to an increase and decrease in SIRT1 and PGC-1α, respectively (Fig. 6E, G and I). However, when treated with SRT1460, Gpbar1 expression showed no significant change despite an increase in SIRT1 expression (Fig. 6F, H and J). From these findings, it can be inferred that Gpbar1 can regulate the SIRT1-PGC-1α signaling pathway, thus establishing Gpbar1 as an upstream regulatory factor of the SIRT1-PGC-1α pathway.

Fig. 6
figure 6

Validation of Gpbar1, SIRT1, PGC-1α upstream and downstream regulation. (A-B) Western blot analysis of SIRT1 and PGC-1α protein expression in HK-2 cells after treatment with EX527 and SR18292 (*p < 0.05, ***p < 0.001). (C-D) qRT-PCR analysis of SIRT1 and PGC-1α mRNA expression in HK-2 cells after treatment with EX527 and SR18292 (***p < 0.001). (E, G) Western blot analysis of protein expression in CON group, Gpbar1 overexpression group, and Gpbar1 knockdown group (*p < 0.05, **p < 0.01, ***p < 0.001). (F, H) Western blot analysis of Gpbar1 and SIRT1 protein expression in HK-2 cells after treatment with SRT1460 (*p < 0.05). (I) qRT-PCR analysis of SIRT1 and PGC-1α mRNA expression in CON group, Gpbar1 overexpression group, and Gpbar1 knockdown group. (J) qRT-PCR analysis of Gpbar1 and SIRT1 mRNA expression in HK-2 cells after treatment with SRT1460 (***p < 0.001).

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Discussion

In this study, we illustrated the role of the Gpbar1-mediated SIRT1-PGC-1α signaling pathway in OJKI and demonstrated the crucial protective effect of Gpbar1-mediated SIRT1-PGC-1α against OJ. The upregulation of Gpbar1 expression enhanced the activity of SIRT1-PGC-1α, thereby reducing oxidative stress, restoring mitochondrial biogenesis, and ultimately protecting against kidney injury.

Deoxycholic acid is the main secondary bile acid retained in cholestasis. In obstructive jaundice, deoxycholic acid cannot be properly eliminated through the enterohepatic circulation and instead stays in various organs, causing organ failure. According to case reports, it can be inferred that renal tubular injury caused by bile and bile salts may be one of the crucial factors leading to acute kidney injury during OJ26. Low concentrations of deoxycholic acid are agonists of Gpbar1, which can promote the expression of Gpbar1 in tissue cells and are beneficial to the body in a variety of diseases. For example, deoxycholic acid inhibits Staphylococcus aureus-induced endometritis by regulating the Gpbar1/PKA/NF-κB signaling pathway27. The widespread distribution of Gpbar1 in the renal tubular epithelium provides histological basis for the concept that stimulating Gpbar1 can protect against bile acid-induced renal injury28. Further studies have shown that Gpbar1 can resist diabetic nephropathy by regulating SIRT1 and SIRT329. However, their study focuses on chronic kidney injury, whereas diabetic nephropathy primarily affects the glomerular basement membrane. Currently, no reports have investigated the protective role of Gpbar1 in acute tubular injury induced by obstructive jaundice. In our study, the observed decrease in Gpbar1, SIRT1, and PGC-1α in the renal tissues of model rats further supports the notion that reduced Gpbar1 activity may be one mechanism underlying renal injury during OJ. Additionally, stimulation of human renal tubular epithelial cells with deoxycholic acid was used to establish a DCA-induced cell injury model, which more realistically simulated the pathophysiological state of renal injury after the occurrence of OJ. In conclusion, our in vitro and in vivo experiments jointly confirmed that excessive bile acid can cause Gpbar1 downregulation, causing the kidney to lose the protective effect of Gpbar1.

OJ, when left unresolved for an extended period, results in prolonged exposure of renal tubules to bile salts and bile acids, leading to a decline in the activities of Gpbar1, SIRT1, and PGC-1α, triggering oxidative stress. The SIRT1-PGC-1α signaling pathway plays a regulatory role in the oxidative stress occurring in tissues. The interaction between SIRT1 and PGC-1α enables deacetylation of PGC-1α, increases its activity, promotes mitochondrial biogenesis, and maintains mitochondrial function30. Similarly, we observed oxidative stress in the renal tissues of model rats and HK-2 cells treated with DCA. Subsequently, we found that overexpression of Gpbar1 increased the activities of SIRT1 and PGC-1α and restored mitochondrial morphology and oxidative stress levels compared to the model group. Moreover, the exacerbation of oxidative stress after Gpbar1 knockdown suggests that maintaining the activity of the Gpbar1-mediated SIRT1-PGC-1α pathway is of great significance in alleviating renal damage during OJ. Increased cellular oxidative stress events can be lethal to cells. In our results, DCA treatment ultimately led to a decline in HK-2 cell viability, while the protective effect of Gpbar1 regulation of oxidative stress was reflected in the impact on cell viability. This protective effect may be achieved through the improvement of cellular energy metabolism, reduction of reactive oxygen species production, and repair of damaged cellular structures, thereby helping to alleviate cellular damage caused by the accumulation of bile salts and bile acids. As a G protein-coupled receptor, Gpbar1 can transmit information through second messengers, such as cAMP. Studies have shown that activating Gpbar1 can partially improve neuronal functional damage through the Gpbar1/cAMP/PKA signaling pathway31. Additionally, in cancer, Gpbar1 is also associated with cAMP; for example, bile acids (BAs) can activate the Gpbar1-cAMP-EGR1 pathway to increase the transcription and expression of FGF19 and FGFR4, promoting the development of gallbladder cancer32. Our study revealed that cAMP levels were altered as detected by ELISA assays. However, whether this is directly related to Gpbar1-mediated SIRT1-PGC-1α signaling requires further experimental investigation.

In summary, this study demonstrates that Gpbar1 can regulate the SIRT1-PGC-1α signaling pathway during OJKI. By preserving mitochondrial biogenesis and protecting against oxidative stress, Gpbar1 helps reduce cellular apoptosis and ultimately alleviates renal damage.