Activation of farnesoid X receptor upregulates binding immunoglobulin protein expression and alleviates diabetic nephropathy

Abstract

BACKGROUND

The exact mechanisms underlying diabetic nephropathy (DN) remain incompletely elucidated, prompting researchers to explore new perspectives and identify novel intervention targets in this field.

AIM

To explore the role and underlying mechanisms of farnesoid X receptor (FXR) in the development of DN by regulating endoplasmic reticulum stress (ERS) molecular chaperone binding immunoglobulin protein (BiP) expression.

METHODS

Bioinformatics analyses identified potential FXR-binding elements in the BiP promoter. Dual-luciferase and chromatin immunoprecipitation (ChIP) assays confirmed FXR-BiP binding sites. In vitro studies used SV40 MES 13 cells under varying glucose conditions and treatments with FXR modulators [obeticholic acid (INT-747) and guggulsterones] or BiP small interfering RNA. The expression of BiP and ERS-related proteins [protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6)] was assessed alongside cell proliferation and extracellular matrix (ECM) synthesis. In vivo studies in DN mice (db/db) examined the effects of FXR activation on renal function and morphology.

RESULTS

FXR bound to the target sequence in the BiP promoter region, enhancing transcriptional activity, as confirmed by ChIP experiments. FXR expression decreased in SV40 MES 13 cells stimulated with high glucose and in renal tissues of DN mice compared with control. Treatment of SV40 MES 13 cells with the FXR agonist INT-747 significantly increased intracellular BiP expression, whereas silencing the FXR gene led to the downregulation of BiP levels. In vivo administration of INT-747 significantly elevated BiP levels in renal tissues, improved renal function and fibrosis in DN mice, while inhibiting the expression of ERS-related signaling proteins PERK, IRE1, and ATF6.

CONCLUSION

FXR promotes BiP expression by binding to its promoter, suppressing ERS pathways, and reducing mesangial cell proliferation and ECM synthesis. These findings highlight FXR as a potential therapeutic target for diabetic glomerulosclerosis.

Key Words: Binding immunoglobulin protein; Chromatin immunoprecipitation; Diabetic nephropathy; Endoplasmic reticulum stress; Farnesoid X receptor

Core Tip: What was known: Farnesoid X receptor (FXR) agonists alleviate proteinuria and glomerulosclerosis in diabetic nephropathy mice by inhibiting inflammation and oxidative stress. Hence, their protective effects have become a research focus in recent years. However, the specific targets and signaling pathways involved in their regulation remain unclear. This study adds: We demonstrated for the first time that using bioinformatics prediction, protein immunoprecipitation, and luciferase reporter assays that FXR directly binds to the promoter region of the binding immunoglobulin protein (BiP) gene. FXR activates the transcriptional activity of BiP, thereby explaining the molecular mechanism of FXR-regulated BiP transcription. We confirmed through in vitro experiments that FXR agonist obeticholic acid (INT-747) administration significantly promotes BiP expression in high-glucose-induced SV40 MES13 cells, while FXR inhibitors downregulated BiP expression. Potential impact: FXR has a direct regulatory effect on BiP. This study provides valuable insights regarding the prevention and treatment of diabetic nephropathy.

INTRODUCTION

Diabetic nephropathy (DN) is one of the most common and harmful microvascular complications of diabetes and a leading cause of end-stage renal disease[1]. The typical pathological manifestations of DN include the phenotypic transformation of glomerular mesangial cells accompanied by extensive synthesis, secretion, and accumulation of the extracellular matrix (ECM), ultimately leading to glomerulosclerosis[2]. However, the exact mechanisms underlying DN remains unclear, prompting researchers to explore new perspectives and identify novel interventional targets.

The endoplasmic reticulum (ER) plays a central role in eukaryotic cells by facilitating the synthesis, proper folding, post-translational modification, and transport of proteins. In recent years, the role of ER stress (ERS) in the pathogenesis of DN has gained increasing attention[3]. Various pathological factors such as hyperglycemia, hypoxia, and oxidative stress can disrupt ER function, causing ERS. Numerous studies have confirmed the involvement of excessive ERS in the development of DN[4-6]. For instance, inhibiting the expression of the negative regulatory protein TRB3 can accelerate proteinuria and renal inflammatory responses in mice with DN[7], whereas blocking the epidermal growth factor receptor can reduce ERS levels and delay DN progression[8]. Additionally, high glucose-induced podocyte apoptosis is partially mediated by ERS[9]. These findings suggest that the effective regulation of ERS may be a critical strategy for the prevention and treatment of DN.

Binding immunoglobulin protein (BiP) is a hallmark molecule and key regulatory factor in ERS[10]. Under normal physiological conditions, BiP acts as a molecular partner. BiP binds to three transmembrane sensor proteins [protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6)] on the ER membrane and renders them inactive. When misfolded or unfolded proteins accumulate in the ER cavity, BiP binds to them and promotes their correct folding, thereby restoring ER homeostasis and enhancing the cell's ability to resist stress[11]. However, under conditions of sustained and intense stress, BiP binds excessively to unfolded proteins, causing the dissociation and activation of PERK, IRE1, and ATF6 signaling factors, which in turn initiate downstream signal transduction and trigger a cascade of reactions, resulting in excessive ERS and cellular dysfunction[12,13]. Previous studies have shown that transgenic mice with downregulated BiP expression exhibit phenotypic transformation of mesangial cells, glomerulosclerosis, and renal tubular fibrosis[14,15], whereas BiP overexpression enhances the resistance of cells to ERS[16]. Research has indicated that the chemical chaperone, 4-phenylbutyric acid (4-PBA), which shares similar biological effects with BiP, can alleviate ERS and inhibit DN development[17,18]. Therefore, moderate upregulation of BiP expression may represent a new target for the prevention and treatment of DN.

The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily. FXR is highly expressed in the kidneys, intestines, and liver[19,20]. Activated FXR forms heterodimers with retinoic acid X receptors, which then regulate gene transcription by binding to FXR response elements in the promoter region of the target gene[21]. Recently, the role of FXR in DN has become an area of growing interest[22,23]. Studies have shown that FXR knockout exacerbates renal damage, whereas FXR agonists alleviate proteinuria and glomerulosclerosis in DN mice by inhibiting inflammation and oxidative stress[24,25]. Given the similar physiological characteristics of FXR agonists, their protective effects against DN have become a research focus in recent years. However, the specific targets and signaling pathways involved in their regulation remain unclear and require further elucidation.

The pathophysiological processes of diabetic kidney disease involve hyperglycemia, inflammation, fibrosis, and hemodynamic alterations. The FXR-BiP pathway may play a significant role in slowing the progression of diabetic kidney disease by regulating metabolism, exerting anti-inflammatory effects, mitigating ER stress, and inhibiting apoptosis. The regulatory interactions between FXR and BiP, as well as their interplay in DN, remains unclear. In this study, we employed bioinformatics analysis to identify a classic binding sequence between FXR and BiP. We then utilized dual-luciferase reporter assays and chromatin immunoprecipitation (ChIP) experiments to validate the interaction between the two. Through cellular experiments, we measured changes in BiP expression in mouse mesangial cells following FXR inhibition or activation, further confirming this regulatory model. We also assessed FXR expression in the renal tissues of db/db mice of different ages and examined the progression of DN following artificial intervention with FXR activity, thereby exploring the impact and significance of FXR regulation on BiP expression in glomerulosclerosis associated with DN.

MATERIALS AND METHODS

Reagents and antibodies

The following antibodies were used in the study: BiP glucose regulated protein 78 (GRP78) antibody (ab21685, Abcam), FXR antibody (ab245624, Abcam), PERK antibody (Cell Signaling Technology), IRE1 antibody (Cell Signaling Technology), PCNA antibody (Cell Signaling Technology), ATF6 antibody (Cell Signaling Technology), fibronectin antibody (Cell Signaling Technology), collagen I antibody (Cell Signaling Technology), α-smooth muscle actin (α-SMA) antibody (Cell Signaling Technology), tubulin antibody (Cell Signaling Technology), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Cell Signaling Technology), horseradish peroxidase (HRP)-labelled goat anti-rabbit IgG antibody (Xavier Biologicals), HRP-labelled goat anti-mouse IgG antibody (Xavier Biologicals), Dylight488-labelled goat anti-rabbit IgG antibody (Abcam), and Dylight594-labelled goat anti-mouse IgG antibody (Abcam).

Cell culture

Mouse glomerular mesangial cells (SV40 MES13) obtained from Wuhan Punosai Life Science and Technology Co. were cultured in Dulbecco’s modified eagle medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin mixture (HyClone), and 1% L-glutamine (HyClone) at 37 °C in a constant temperature incubator (Thermo Scientific) containing 5% carbon dioxide.

Small interfering RNA cell transfection

SV40 MES13 cells were transfected with FXR small interfering RNA (siRNA)-1/siRNA2/siRNA3 obtained from Gemma Genetics Co., Ltd. Transfection was performed using the Lipofectamine 3000 transfection kit (Thermo Fisher Scientific). Cells were collected for polymerase chain reaction (PCR), western blotting (WB), and immunofluorescence analysis 24-48 hours after transfection.

Western blot

Total protein was extracted from SV40 MES13 cells using T-PER protein lysate (Thermo Fisher Scientific) following the manufacturer’s instructions. Protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. Primary antibodies used included GRP78, FXR, fibronectin, collagen II, α-SMA, and GAPDH. Enhanced chemiluminescence WB substrate (Thermo Fisher Scientific) was used for visualization and ImageJ-win64 software was used for band analysis.

Quantitative reverse transcription PCR

Total RNA was extracted from SV40 MES13 cells using RNAiso Plus according to the manufacturer’s instructions. Complementary DNA was synthesized via reverse transcription using the PrimeScript RT kit (TaKaRa Biotechnology). GAPDH served as the reference gene for normalization. Quantitative real-time PCR (qRT-PCR) was conducted on an ABI ViiA7DX system utilizing SYBR Premix Ex Taq II (TaKaRa) for amplification and analysis.

Immunofluorescence and immunocytochemistry

The cells were treated using Triton X-100 Cell Lysate (Shanghai Sunbow Biotechnology Co., Ltd.) following the manufacturer’s instructions. After incubation with primary antibodies (GRP78, FXR, fibronectin, collagen II, α-SMA, and GAPDH), fluorescently labelled secondary antibodies were applied, and images were captured using a fluorescence microscope.

ChIP

ChIP experiments were conducted according to the instructions of the QuickChIP kit (IMGENEX). This process involves protein and DNA cross-linking, genomic DNA shearing, immunoprecipitation, capture of triplet complexes, decrosslinking, and DNA analysis to determine the binding of FXR to specific sequences in the promoter region of BiP.

Animal models

Male db/m and db/db mice were obtained from Spearfish Biotechnology Co. The mice were placed in specific pathogen free animal facilities and the experiments were conducted in accordance with ethical guidelines. Mice were treated with the FXR agonist obeticholic acid (INT-747) and kidney samples were collected for further analysis.

Randomly divide db/db mice into 3 groups (n = 8): Negative control group (CON), INT-747 group, guggulsterone group, Db/m mice (n = 8) serve as the positive control group. Drug administration begins at week 8, with the specific protocol as follows: Db/m group: No drug administration. Db/db (CON) group: Intravenous tail injection of solvent [10% dimethyl sulfoxide (DMSO) + 40% polyethylene glycol 300 (PEG300) + 45% saline + 5% tween 80] every 3 days, for a total of 6 times. Db/db (INT-747) group: Intravenous tail injection of INT-747 (10 mg/kg, dissolved in 10% DMSO + 40% PEG300 + 45% saline + 5% tween 80) every 3 days, for a total of 6 times. Db/db (guggulsterone) group: Intravenous tail injection of guggulsterone (100 mg/kg, dissolved in 10% DMSO + 40% PEG300 + 45% saline + 5% tween 80) every 3 days, for a total of 6 times. Drug administration continues for 18 days.

The study protocol was approved by the ethical committee of the University-Town Hospital of Chongqing Medical University with approval No. LL-202127.

Histological staining and immunohistochemistry

Sections from fixed kidney samples were subjected to hematoxylin and eosin staining and immunohistochemistry using specific antibodies. Images were captured and analyzed using the ImageJ software.

Statistical analysis

Data are presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism 10 software. To analyze intergroup differences, an unpaired Student’s t-test was employed, as this study involved comparisons across multiple groups. This statistical method was utilized to assess the significance of differences observed between the groups. Unpaired Student’s t-test was used to determine statistical significance.

RESULTS

FXR positively regulates its expression by binding to the BiP promoter region

To elucidate the molecular mechanism by which FXR regulates BiP expression, we first used bioinformatics methods to analyze and predict the possible binding sites of FXR in the promoter region of the human BiP gene. The predicted results are listed in Table 1. Comparison of the predicted results with the classic FXR-binding sequence IR1 (sequence naming IR1) (AGGTCAnTGACCT) revealed that DR2 (sequence naming DR2) (1374, analysis score 0.928) in the BiP promoter region was the most likely FXR-binding site.

Table 1 Farnesoid X receptor potential binding site sequence and score of human binding immunoglobulin protein gene promoter region (farnesoid X receptor binding site analysis software).

Classic sequence	Position of positive and negative chain sequence (bp)	Score	Z score	P value	E value	Half point E value	Binding site sequence (5’-3’)
DR2	1374 (-)	0.928		0.0006372			GGGTCAGGAGATCA
DR6	376 (-)	0.824		0.0084952			GGATCACGAGGTCA
DR4	1382 (-)	0.802		0.0140067			GGATCACGGGGTCA
IR8	66 (-)	0.765		0.0170365			GGGTCACTCCTGGGACCT
DR1	2159 (-)	0.727		0.0287146			GGGGCAAAGGTTA
DR8	750 (-)	0.716		0.0254571			GGGTCAGAAGTCAGGAGA

To validate these findings, mesangial cells were transfected with PGL3 plasmid containing the full-length BiP promoter region, and the effects of FXR activation on BiP transcription were analyzed. BiP luciferase activity increased in a dose-dependent manner after INT-747 treatment (Figure 1A). To pinpoint the binding site within the promoter, truncated promoter fragments were tested. Plasmids containing a 376-bp fragment upstream of the transcription start site exhibited a significant increase in luciferase activity, comparable to the full-length promoter. In contrast, a + 200 bp fragment showed a similar signal to the full-length construct (Figure 1B). These results indicate that the FXR-binding site likely resides between - 376 bp and + 200 bp of the BiP promoter.

Figure 1 Farnesoid X receptor positively regulates its expression by binding to the binding immunoglobulin protein promoter region. A: Fluorescent enzyme reporter gene detection showed that the activity of the binding immunoglobulin protein (BiP) gene promoter increased in a dose-dependent manner after treatment with farnesoid X receptor agonist obeticholic acid (INT-747); B: After truncating the BiP gene promoter sequence, luciferase reporter gene detection showed that transcriptional activity was still present in the -376 to +200 sequence of the BiP gene promoter; C: Chromatin immunoprecipitation quantitative real time polymerase chain reaction results show that anti farnesoid X receptor antibodies significantly enrich the promoter sequence of BiP gene. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. HG: High glucose; DMSO: Dimethyl sulfoxide; INT-747: Obeticholic acid; FXR: Farnesoid X receptor; Luc: Luciferase.

Subsequently, ChIP qRT-PCR experiments showed that antibodies against FXR enriched the promoter sequence of BiP (Figure 1C), indicating an interaction between FXR and the BiP promoter.

Downregulation of FXR expression in mesangial cells under high glucose conditions

First, we compared the effects of different sugar concentrations on FXR expression in mouse glomerular mesangial cells. RT-qPCR and WB results showed that after high glucose treatment, the expression of FXR message RNA and protein decreased in a dose-dependent manner (Figure 2A and B), indicating insufficient expression and secretion of FXR in mesangial cells under high glucose conditions. Immunocytochemical staining confirmed that high glucose treatment decreased FXR expression (Figure 2C).

Figure 2 Downregulation of farnesoid X receptor expression in mesangial cells under high glucose conditions, farnesoid X receptor has a positive regulatory effect on binding immunoglobulin protein expression in mesangial cells. A: Bar chart shows that the message RNA level of farnesoid X receptor (FXR) decreases with increasing glucose concentration, as detected by quantitative real-time polymerase chain reaction; B: Western blot shows that the protein level of FXR decreases with increasing glucose concentration; C: Immunocytochemical staining detected a decrease in FXR expression in high glucose-treated mesangial cells compared to the control group; D: Western blot detection shows a synchronous decrease in binding immunoglobulin protein (BiP) expression after small interfering RNA inhibition of FXR; E: Western blot detection of the effect of different concentrations of FXR agonists or inhibitors on BiP protein levels in mesangial cells. ^bP < 0.01. ^cP < 0.001. HG: High glucose; INT-747: Obeticholic acid; FXR: Farnesoid X receptor; NC: Negative control; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; DAB: 3,3’-diaminobenzidine color development; BiP: Binding immunoglobulin protein; TUBLIN: Tubulin antibody.

FXR has a positive regulatory effect on BIP expression in mesangial cells

To confirm the regulatory relationship between FXR and BIP, we applied siRNA to suppress FXR in mesangial cells, resulting in a synchronous decrease in BIP expression (Figure 2D). BIP expression increased in a dose-dependent manner following treatment with the FXR agonist INT-747. After the addition of FXR inhibitors, the expression of BiP decreased, confirming that FXR has a previous reports have indicated regulatory effect on BiP expression (Figure 2E).

FXR regulates the effect of BIP on downstream ERS-related signaling molecules

To further verify whether FXR inhibits the downstream ERS signaling pathway by promoting BiP expression, we treated mesangial cells with the FXR agonist INT-747 and found downregulation of the ERS-related signaling molecules PERK, IRE1, and ATF6 through qRT-PCR and WB analysis. However, after treatment with the FXR inhibitor guggulsterone or siRNA to inhibit BiP expression, upregulated PERK, IRE1, and ATF6 expressions were observed. When INT-747 and siRNA were added simultaneously to inhibit BiP, PERK, IRE1, and ATF6 expressions were not significantly downregulated (Figure 3). These findings suggest that the overexpression of FXR significantly inhibits the expression of ERS-related signaling proteins, and this effect is achieved through BiP upregulation.

Figure 3 Farnesoid X receptor regulates the effect of binding immunoglobulin protein on downstream endoplasmic reticulum stress related signaling molecules. A: Bar chart showing the expression levels of protein kinase R-like endoplasmic reticulum kinase (PERK) message RNA (mRNA) in different treatment groups, detected by quantitative real-time polymerase chain reaction (qRT-PCR); B: Western blot (WB) displays the expression levels of PERK protein in different treatment groups, and the bar chart shows the grayscale values of WB bands; C: Bar chart showing the expression levels of inositol-requiring enzyme 1 (IRE1) mRNA in different treatment groups, detected by qRT-PCR; D: WB displays the expression levels of the IRE1 protein in different treatment groups, with the bar chart showing the grayscale values of WB bands; E: Bar chart showing the expression levels of activating transcription factor 6 (ATF6) mRNA in different treatment groups, detected by qRT-PCR; F: WB displays the expression levels of the ATF6 protein in different treatment groups, with the bar chart showing the grayscale values of WB bands. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. HG: High glucose; LG: Low glucose; INT-747: Obeticholic acid; BiP: Binding immunoglobulin protein; Gugg: Guggulsterones; siRNA: Small interfering RNA; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; PERK: Protein kinase R-like endoplasmic reticulum kinase; IRE1: Inositol-requiring enzyme 1; ATF6: Activating transcription factor 6.

Effects of artificial intervention on FXR activity on mesangial cell proliferation and ECM synthesis

After high glucose treatment, the proliferation of mesangial cells increased significantly. After treatment with INT-747, the proliferation of mesangial cells decreased significantly, whereas after treatment with guggulsterone, the proliferation of mesangial cells was significantly upregulated (Figure 4A). PCNA is a nuclear marker of cell proliferation, including mesangial cell proliferation. We detected the PCNA value of mesangial cells by WB and confirmed that after treatment with FXR agonists, both mesangial cell proliferation and PCNA expression reduced significantly (Figure 4B). WB detection of ECM synthesis confirmed that FXR agonists downregulated the expression of fibrosis related proteins such as α-SMA, fibronectin, and collagen, while FXR inhibitors had the opposite effect (Figure 4C-E). These results indicate that FXR activation inhibits mesangial cell proliferation and ECM synthesis.

Figure 4 The effect of artificial intervention on farnesoid X receptor activity on mesangial cell proliferation and extracellular matrix synthesis. A: Cell counting kit-8 detection of mesangial cell proliferation; B-E: Western blot detection of the expression of PCNA, fibronectin, α-smooth muscle actin, and collagen I. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. HG: High glucose; LG: Low glucose; INT-747: Obeticholic acid; Gugg: Guggulsterones; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; OD: Optical density; α-SMA: α-smooth muscle actin.

FXR agonist upregulates BiP expression in db/db mice and improves renal fibrosis in DN mice

After confirming the downregulation of FXR expression in DN mouse kidneys, we examined the changes in DN mouse kidney tissue after treatment with the FXR agonist INT-747 and the inhibitor guggulsterone. The results showed that INT-747 treatment improved renal fibrosis in DN mice (Figure 5A). Meanwhile, FXR activation also downregulated the expression of fibrosis-related proteins such as α-SMA, fibronectin, and collagen I (Figure 5B), demonstrating the renal protective effect of FXR.

Figure 5 Farnesoid X receptor agonist upregulates binding immunoglobulin protein expression in db/db mice and improves renal fibrosis in diabetic nephropathy mice. A: Pathological changes in mouse kidneys after treatment with farnesoid X receptor (FXR) agonists and inhibitors; B: Immunohistochemical detection of the effects of FXR agonists and inhibitors on the expression of renal fibrosis related proteins. NC: Negative control; HE: Hematoxylin eosin; PAS: Periodic acid-Schiff stain; INT-747: Obeticholic acid; α-SMA: α-smooth muscle actin.

Downregulation of FXR expression in renal tissue of DN mice

The immunohistochemical results showed that the expression of FXR protein in the renal tissue of db/db mice gradually decreased with increasing age, compared with the db/m control group (Figure 6A).

Figure 6 Downregulation of farnesoid X receptor expression in diabetic nephropathy mouse kidney tissue, changes in serum biochemical indicators of mice after farnesoid X receptor agonist intervention. A: Immunohistochemical staining detection of farnesoid X receptor expression in renal tissue of db/db mice at different weeks of age compared to db/m; B: Changes in blood creatinine levels in db/m and db/db mice at different weeks of age; C-E: Changes in urinary and serum biochemical indicators in db/db mice treated with obeticholic acid. NC: Negative control; INT-747: Obeticholic acid; Scr: Serum creatinine; UACR: Urine albumin-to-creatinine ratio; CR: Creatinine; BUN: Blood urea nitrogen.

Changes in serum biochemical indicators of mice after FXR agonist intervention

Biochemical tests confirmed that db/db mice developed kidney lesions at week 10 (Figure 6B). Therefore, INT-747 was injected into db/db mice at week 10. After six weeks of treatment with INT-747, the db/db (INT-747) group showed a decrease in serum creatinine and a reduction in the urinary microalbumin excretion rate after INT-747 treatment, indicating an improvement in renal function after intervention with the FXR agonist INT-747 (Figure 6C-E).

DISCUSSION

This study revealed the protective role and underlying mechanisms of the transcription factor FXR in the pathogenesis of DN by promoting the expression of the ERS chaperone molecular BiP through in vivo and in vitro experiments.

First, through bioinformatics prediction, ChIP, and luciferase reporter assays, we demonstrated for the first time that FXR can directly bind to the promoter region of the BiP gene and activate its transcriptional activity, elucidating the molecular mechanism by which FXR positively regulates BiP transcription. FXR is known to transcriptionally repress CYP7A1, the rate-limiting enzyme in bile acid synthesis[26,27]. However, its role in regulating ERS-related gene transcription remains largely unexplored. Moreover, the ERS response pathway involves multiple signaling branches, including BiP, PERK, IRE1α, and ATF6[28], with BiP serving as the most upstream molecular chaperone in sensing ERS and initiating downstream cascades. Therefore, elucidating the specific mechanism by which FXR upregulates BiP transcription is vital for understanding the core mechanisms by which FXR regulates ERS.

Previous studies have confirmed that BiP overexpression via transgenic approaches significantly enhances the ERS response and initiates cellular self-protective effects[29]. Conversely, mutation and downregulation of the BiP gene through genetic recombination lead to the phenotypic transformation of mesangial cells, glomerulosclerosis, and renal tubular fibrosis in transgenic mice[30,31]. Related studies have also shown that the exogenous chemical chaperone 4-PBA can alleviate ERS and inhibit DN progression, suggesting that upregulating endogenous molecular chaperone BiP expression may be a new target for DN prevention and treatment.

In DN mouse models and high glucose-induced mouse mesangial cells, FXR expression was significantly reduced, indicating that ERS activation and FXR dysregulation may be critical pathological foundations for DN onset. Numerous studies on diabetes and its complications have confirmed that sustained activation of the ERS pathway is a key factor leading to diabetes-related damage to tissues and organs[32-34]. For example, Liu et al[35] detected the significantly elevated expression of ERS markers, such as BiP, in renal biopsy specimens from patients with DN, suggesting a close relationship between ERS and DN. As a member of the nuclear receptor superfamily enriched in the liver and kidneys, FXR plays an important role in regulating bile acid, lipid, and glucose homeostasis[36,37]. Previous studies have shown that the FXR agonist INT-747 can ameliorate renal damage in type 2 diabetic mice. Since this agonist is a synthetic bile acid derivative, it has low toxicity and high safety in vivo[38,39]. However, its specific in vivo mechanisms remain unclear. Our findings further revealed that the downregulation of FXR expression may serve as an upstream initiator of ERS activation and the onset of DN.

INT-747, a potent and selective FXR agonist, demonstrates significant biological activity through direct activation of FXR. This activation enables the regulation of multiple critical physiological processes, including bile acid homeostasis, lipid metabolism, glucose metabolism, and inflammatory responses. In contrast, guggulsterone, a well-characterized FXR antagonist, has been extensively investigated for its dual role in modulating lipid metabolism and inflammatory pathways, making it a valuable pharmacological tool for studying FXR inhibition mechanisms.

Subsequently, we confirmed through in vitro experiments that administration of the FXR agonist INT-747 significantly promoted BiP expression in high glucose-induced SV40 MES13 cells, whereas FXR inhibitors downregulated BiP expression, indicating that FXR has a direct regulatory effect on BiP. BiP, also referred to as GRP78, is an ER stress response protein. Its expression is tightly regulated by multiple transcription factors, including ATF6 and X-box binding protein 1[40]. Currently, research on the mechanism of FXR action in renal cells is limited. Wang et al[41] reported that FXR agonists alleviate inflammation and apoptosis in renal tubular epithelial cells by inhibiting the nuclear factor kappa-B pathway. Meanwhile, Yao et al[42] found that FXR activation can reduce high glucose-induced damage in mesangial cells by upregulating the expression of antioxidant genes such as Nrf2. This study is the first to reveal, in the context of DN, the direct upregulation effect of FXR on BiP, a key effector molecule of ERS, thereby expanding our understanding of the new mechanisms of FXR renal protection.

Finally, this study further validates the inhibitory effect of FXR activation on DN progression through in vivo experiments. Administration of the FXR agonist INT-747 significantly improved renal function and pathological damage in DN mice while promoting BiP expression, suggesting that the upregulation of FXR expression may be an effective strategy to delay DN. Notably, FXR activation not only inhibited the expression of ERS signaling proteins, but also significantly improved clinical indicators such as proteinuria, glomerulosclerosis, and renal interstitial fibrosis in DN mice. This suggests that the BiP-ERS pathway may be the core effector pathway for the renoprotective effects of FXR. Previous studies have indicated that sustained ERS activation induces various downstream effects, including inflammatory responses, mesangial cell proliferation, and ECM deposition, ultimately leading to irreversible kidney structural and functional damage[43-47]. Conversely, blocking ERS can significantly alleviate proteinuria, glomerulosclerosis, and renal interstitial fibrosis in DN mice and delay the onset of renal failure[48,49]. This study confirmed that FXR agonists can mitigate the cascading effects induced by ER imbalance by promoting the BiP-ERS signaling axis, thereby comprehensively improving renal prognosis in DN mice. This aligns with multifaceted roles of FXR in regulating metabolic homeostasis and organ fibrosis[50,51], highlighting FXR as an ideal drug target for the prevention and treatment of DN.

In conclusion, this study elucidated the protective role and mechanisms of FXR in the progression of DN by promoting BiP expression at the transcriptional level and alleviating kidney damage induced by ERS, providing new insights and strategies for the prevention and treatment of ERS-related DN.

CONCLUSION

In conclusion, this study elucidated the protective role and mechanisms of FXR in the progression of DN by promoting BiP expression at the transcriptional level and alleviating kidney damage induced by ERS, providing new insights and strategies for the prevention and treatment of ERS-related DN.

ACKNOWLEDGEMENTS

We acknowledge support of the Mu’s research group.