C57BL/6J mice, weighing 22–25 g and aged 7–8 weeks, were obtained from Jinan Pengyue Laboratory Animal Breeding Co., Ltd. Nrf2-knockout (Nrf2-KO) mice with a C57BL/6J background were purchased from Jiangsu Huachuang Sino Pharma Co. The pentobarbital-isoflurane anesthesia protocol was used in our animal experiments. To establish the I/R injury model, atraumatic vascular clips were used to clamp the bilateral renal pedicles of mice for 30 min, followed by reperfusion for 24 h (Lee et al., 2020). After euthanasia, blood and renal tissues were collected immediately for further analysis.

To assess the renoprotective effect of 5-MTP, the mice were randomly assigned to four groups (n = 5). In the Sham and 5-MTP groups, renal pedicles were exposed but not subjected to clamping. The mice in the designed groups were administered an intraperitoneal injection (100 mg/kg) of 5-MTP (M4001, Sigma, United States) 30 min before the operation. The mice in the I/R group received an equal volume of saline. Additionally, to examine the association between the Nrf2/HO-1 pathway and the renoprotective effects of 5-MTP, wild-type (WT) or Nrf2-KO mice were randomized into three groups, each containing five mice.

Renal tissues were embedded in paraffin, cut into slices (4-μm-thick), and stained with hematoxylin-eosin (HE). Two experienced pathologists independently evaluated histopathological changes in the renal tissues. As described in previous studies, the tubular injury score, which ranges from 0 to 4, is used to assess the severity of renal tissue damage (Yan et al., 2020).

After collecting blood samples from the mice, the samples were left undisturbed at room temperature for 20 min. Next, the samples were centrifuged at 2,500 revolutions per minute (rpm)for 15 min at 4°C. The upper layer of serum was removed and stored at −20°C. The concentrations of blood urea nitrogen (BUN) and serum creatinine (SCr) were measured using an automated biochemical analyzer for animals (BS-240Vet, Mindray, China), which served as indicators to evaluate renal function in the mice.

The concentrations of IL-1β and IL-6 in mice serum were estimated using commercial ELISA kits (Solarbio, China). Following the manufacturer’s guidelines, absorbance readings were obtained at 450 nm and 630 nm, and the concentrations were subsequently calculated based on standard curves.

The total and oxidized glutathione levels were evaluated using a GSSG/GSH quantification kit (Jiancheng, China). The GSH/GSSG ratio was calculated. The total antioxidant capacity (T-AOC) was assessed by a T-AOC assay kit that uses the rapid ABTS method (Beyotime, China).

The expression level of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) was assessed by IF staining. After fixation with paraformaldehyde, tissue sections were washed three times with phosphate-buffered saline (PBS) and incubated overnight with an anti-CHOP antibody (CST #2895, United States). Next, the sections were incubated with a fluorescently labeled secondary antibody for 1 h. After incubation, 4,6-diamidino-2-phenylindole (DAPI) was used to visualize nuclei. IF intensity was quantitatively analyzed using the ImageJ software (NIH, United States).

TUNEL staining was conducted using a TUNEL Kit (Roche, United States). First, the sections were deparaffinized and treated with proteinase K for 10 min. Then, TUNEL labeling buffer was added. These sections were subsequently maintained in a humidified, light-tight container at 37°C for 1 h. The cell nuclei were stained with hematoxylin for 10 min.

The Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, China) was used for nuclear and cytoplasmic proteins extraction. Total proteins were extracted using a total protein isolation kit (Beyotime, China). Protein concentrations were assessed using a bicinchoninic acid (BCA) assay kit (Beyotime, China). SDS-PAGE (10% or 12%) was performed to separate the proteins. After electrophoresis, the separated proteins were transferred to polyvinylidene difluoride membranes. The primary antibodies against ATF4 (1:1,000, CST #11815, United States), ATF6 (1:1,000, CST #65880, United States), CHOP (1:1,000, CST #2895, United States), DR5 (1:1,000, Bioss, China), eIF2α (1:1000, Affinity AF6087, United States), HO-1 (1:2,000, CST #43966, United States), IRE1α (1:1000, Affinity DF7709, United States), Nrf2 (1:1,000, CST #12721, United States), p-IRE1α (1:1,000, Affinity AF7150, United States), PERK (1:1,000, Affinity AF5304, United States), p-eIF2α (1:1,000, Affinity AF3087, United States), p-PERK (1:1,000, Affinity DF7576, United States), β-actin (1:1,000, Affinity AF7018, United States) and GAPDH (1:1,000, Affinity AF7021, United States) were incubated overnight. The corresponding secondary antibodies were subsequently incubated for 1 h, and the blots were analyzed using the ImageJ software (NIH, United States).

RNA was extracted using TRIzol reagent (Invitrogen, United States). The PrimeScript RT Master Mix (Takara, Japan) was used to synthesize cDNA. The qRT-PCR procedure was performed using SYBR Green qPCR master mix (Takara, Japan); the primer sequences used are listed in Supplementary Table S1.

We obtained the GSE212678 dataset from the GEO database and subsequently identified the differentially expressed genes (DEGs) between I/R and sham mice (Huang et al., 2024). GO and KEGG enrichment analyses were performed on the DEGs (Supplementary Table S2). We obtained Structure Data File (SDF) and Simplified Molecular Input Line Entry System (SMILES) files for the 5-MTP from PubChem (https://pubchem.ncbi.nlm.nih.gov/). The therapeutic targets of 5-MTP in renal I/R injury were predicted using the SwissTargetPrediction (Daina et al., 2019), PharmMapper (Wang et al., 2017), BindingDB (https://www.bindingdb.org), and GeneCards (https://www.genecards.org/) databases. A protein-protein interaction (PPI) network was constructed using the STRING database (https://www.string-db.org/), followed by visualization with Cytoscape (v3.9.0). Additionally, the key factors were identified with the cytoHubba in Cytoscape.

HK-2 cells were obtained from the Cell Bank of the Chinese Academy of Sciences. DMEM and fetal bovine serum were purchased from Gibco (United States). A hypoxic environment was mimicked using a three-gas incubator with 1% O₂, 94% N₂, and 5% CO_2, and the cells were maintained in a hypoxic environment for 24 h. Next, the cells were cultured with a fresh medium under normoxic conditions for 6 h. Before exposure to hypoxia/reoxygenation (H/R) treatment, the cells were treated with various doses of 5-MTP dissolved in dimethyl sulfoxide (DMSO) for 12 h. The cells were then divided into five groups.

Custom-designed Nrf2 siRNA and negative control (NC) siRNA were purchased from Shanghai GenePharma Co., Ltd. (China). The cells were efficiently transfected with these siRNAs using Lipofectamine 3000 (Invitrogen, United States). The qRT-PCR analysis was conducted to assess the efficacy of knocking down Nrf2 after transfection for 24 h.

To assess the viability of HK-2 cells subjected to H/R, the Cell Counting Kit-8 (CCK-8) assay (Beyotime, China) was performed. The cells were transferred to a 96-well plate. Each well received 10 μL of CCK8 solution and was incubated for 2.5 h at standard temperature. The absorbance was recorded at 450 nm.

The cell apoptosis rate was assessed using an Annexin V-FITC/PI Apoptosis Kit (Elabscience, China). HK-2 cells were seeded at a density of 1 × 10⁵ cells/well in six-well plates. Following incubation for 24 h, modeling and drug administration were conducted as previously described, and the cells were subsequently collected for further procedures. After centrifugation, the cells were rinsed and resuspended in a binding buffer. Next, the cells were incubated with Annexin V-FITC and PI reagents for 15 min. Finally, flow cytometry was performed using a BD flow cytometer (United States).

The data are presented as the mean ± standard deviation. The differences among and between groups were determined by one-way or two-way ANOVA with post hoc Bonferroni test using the Graph Prism 9 software (GraphPad Software, United States). All differences were considered to be statistically significant at P < 0.05.

To evaluate the renoprotective effect of 5-MTP pretreatment on renal I/R injury, histopathological analysis was performed, and the tubular injury score was assessed by HE staining. Compared to the Sham group, the I/R group presented severe pathological damage, specifically characterized by tubular necrosis, dilatation, edema, inflammatory infiltration, and cast formation. Notably, 5-MTP pretreatment significantly attenuated these pathological changes (Figure 1A). Similarly, the tubular injury score was considerably higher in the I/R group than in the Sham group. This score was considerably lower in the I/R+5-MTP group than in the I/R group (Figure 1B).

Renal function was assessed by detecting the BUN and SCr levels (Figures 1C,D). The I/R group presented higher BUN and SCr levels, indicating impaired renal function. However, 5-MTP pretreatment substantially improved renal function. To further investigate the level of inflammation, we measured the IL-1β and IL-6 levels (Figures 1E,F). I/R injury significantly increased the serum IL-1β and IL-6 levels; however, 5-MTP suppressed the secretion of these inflammatory cytokines. Additionally, to evaluate the levels of oxidative stress in the kidney, we detected the GSH/GSSG ratio and T-AOC (Figures 1G,H). I/R injury reduced the GSH/GSSG ratio and T-AOC, indicating an increase in oxidative stress, which was mitigated by 5-MTP pretreatment. We also detected the mRNA expression levels of Neutrophil Gelatinase-Associated Lipocalin (NGAL) and Kidney Injury Molecule-1 (KIM-1), which are markers of renal injury, by conducting qRT-PCR (Figures 1I,J). I/R injury significantly increased NGAL and KIM-1 mRNA levels, indicating renal proximal tubular damage. However, 5-MTP pretreatment significantly attenuated these changes. Our findings indicated that 5-MTP pretreatment exerts renoprotective effects, particularly by alleviating renal tissue injury, improving renal function, and mitigating inflammatory responses and oxidative stress.

To evaluate the level of ERS-mediated apoptosis in renal I/R injury, we initially used bioinformatics methods. Specifically, the DEGs between sham and renal I/R mice in the GSE212678 dataset were analyzed and 3042 DEGs were identified, which included 1,713 upregulated and 1,329 downregulated genes (|log₂FC| > 0.58, P.adj < 0.05) (Figure 2A). The mRNA expression levels of ATF4 and DR5, which are both crucial factors in ERS-mediated apoptosis, increased significantly, indicating the activation of ERS-mediated apoptosis in I/R model mice. By conducting GO and KEGG analyses, we found that the DEGs are involved primarily in the regulation of apoptotic cell clearance and the endoplasmic reticulum lumen (Figure 2B). To elucidate the results of the bioinformatics analysis, WB assays were performed to determine whether 5-MTP attenuated ERS and ERS-mediated apoptosis caused by renal I/R injury (Figures 2C–H). We assessed the level of ERS by determining the expression levels of ATF6, p-PERK, PERK, IRE1α and p-IRE1α through WB analysis. These results indicated that I/R injury considerably increased the protein expression level of ATF6 and the phosphorylation levels of PERK and IRE1α. However, pretreatment with 5-MTP partially decreased this effect. Moreover, renal I/R injury markedly increased the expression of ATF4, CHOP, and DR5, along with eIF2α phosphorylation level, whereas 5-MTP mitigated these changes. The IF results revealed that I/R injury greatly increased CHOP expression, whereas 5-MTP mitigated this change (Figures 2I,K). Apoptosis plays a key role in exacerbating various diseases caused by ERS (Zhang et al., 2022a). Apoptosis in kidney tissues was assessed by TUNEL staining (Figures 2J,L). The I/R group presented a significantly greater number of TUNEL-positive cells. Notably, 5-MTP greatly alleviated apoptosis in the I/R group. These findings indicates that 5-MTP can alleviate ERS and ERS-mediated apoptosis in mice with I/R.

To investigate the molecular mechanisms underlying the renoprotective effect of 5-MTP, the SwissTargetPrediction, PharmMapper, and BindingDB databases were used to identify 482 potential targets of 5-MTP (13 duplicate targets eliminated). Next, genes related to renal I/R injury were retrieved from the GeneCards database, and Venn diagram analysis was performed to identify 60 overlapping genes (Figure 3A). To elucidate the primary target of 5-MTP, a PPI network was constructed using the STRING database (Figure 3B). The BottleNeck algorithm in Cytohubba was used to evaluate the correlations among the targets (Figure 3C). We found that NFE2L2 exhibited a strong correlation. Another study revealed that 5-MTP can enhance acute lung injury recovery by activating the Nrf2/HO-1 pathway (Ma et al., 2023). We performed WB analysis to confirm the activation of the Nrf2/HO-1 pathway by 5-MTP in renal I/R injury (Figures 3D–G). The results revealed that 5-MTP increases both total Nrf2 expression levels and nuclear translocation of Nrf2, accompanied by an elevation in HO-1 protein expression. These findings confirm 5-MTP can activate the Nrf2/HO-1 pathway in renal I/R injury.

The Nrf2 gene was silenced in HK2 cells via siRNA. Using these Nrf2-knockdown cells, we confirmed the association between the Nrf2/HO-1 pathway and the amelioration of H/R injury by 5-MTP. The CCK-8 assay was conducted to assess the viability of HK-2 cells subjected to H/R and the effect of 5-MTP treatment at different concentrations. Before H/R exposure, the cells were incubated with 5, 15, 25, 50, or 100 μM 5-MTP for 12 h. Dose-dependent protection was observed at 5–50 μM (Figure 4A). Additionally, no significant difference in cell viability was observed between the doses of 100 and 50 μM (P > 0.05). Therefore, 50 μM 5-MTP was selected for use in subsequent experiments. The efficacy of Nrf2 knockdown was conformed by qRT-PCR (Figure 4B). As shown in Figure 4C, 5-MTP could ameliorate the damage caused by H/R. However, this cytoprotective effect of 5-MTP was notably inhibited in Nrf2-silenced cells. To assess the anti-inflammatory effect of 5-MTP in vitro, we measured the IL-1β and IL-6 levels. We found that 5-MTP significantly decreased the concentrations of both cytokines. However, silencing Nrf2 inhibited the anti-inflammatory effects of 5-MTP (Figures 4D,E). Additionally, based on the T-AOC levels and the GSH/GSSG ratio, 5-MTP showed the ability to mitigate oxidative stress, whereas knocking down Nrf2 significantly attenuated the antioxidant effect of 5-MTP (Figures 4F,G). As shown in Figures 4P–S, treatment with 5-MTP significantly increased the nuclear-to-cytoplasmic ratio of Nrf2, supporting active nuclear translocation. Concurrently, cytoplasmic Nrf2 levels exhibited a moderate elevation, suggesting that 5-MTP may also enhance total Nrf2 protein stability or synthesis. These findings collectively demonstrate that 5-MTP activates the Nrf2/HO-1 pathway through a dual mechanism involving nuclear translocation and potential modulation of Nrf2 protein levels. Consistent with Nrf2 activation, 5-MTP treatment robustly increased HO-1 expression. To evaluate the role of 5-MTP in ERS-mediated apoptosis in vitro, we conducted WB analysis to examine the expression levels of proteins related to ERS and ERS-mediated apoptosis (Figures 4H–O). H/R significantly increased the levels of ATF6, ATF4, CHOP, and DR5, as well as the levels of phosphorylated PERK, IRE1α, and eIF2α. However, 5-MTP effectively reversed this change, indicating its ability to mitigate H/R-induced ERS-mediated apoptosis. This effect of 5-MTP was not observed in the siNrf2+H/R+5-MTP group. Flow cytometry was then conducted to assess the early apoptosis, and the results revealed that 5-MTP significantly inhibited H/R-induced apoptosis (Figure 4T). However, this effect was also significantly inhibited in cells in which Nrf2 was knocked down. Our in vitro studies reveal that 5-MTP can mitigate ERS-mediated apoptosis by activating the Nrf2/HO-1 pathway.

To evaluate the role of the Nrf2/HO-1 pathway in mediating the renoprotective effects of 5-MTP in vivo, we compared outcomes between WT and Nrf2-KO mice. Compared to WT mice, Nrf2-KO mice subjected to I/R and 5-MTP presented more severe renal injury and much higher tubular injury scores (Figures 5A,B). By assessing SCr and BUN, we found that the improvement in renal function decline caused by 5-MTP in renal I/R injury was substantially suppressed in Nrf2-KO mice (Figures 5C,D). We also assessed whether the anti-inflammatory and antioxidant abilities were suppressed in Nrf2-KO mice (Figures 5E–H). Compared to WT mice, Nrf2-KO mice subjected to I/R and 5-MTP presented markedly higher levels of IL-1β and IL-6 and significantly lower GSH/GSSG ratios and T-AOC levels. Moreover, a considerable increase in NGAL and KIM-1 mRNAs indicated a significant attenuation of the efficacy of 5-MTP in mitigating kidney injury in Nrf2-KO mice (Figures 5I,J). These results suggest that the therapeutic efficacy of 5-MTP in I/R injury is strongly associated with the Nrf2/HO-1 pathway.

We subsequently assessed the expression of relevant proteins in vivo to confirm the role of the Nrf2/HO-1 pathway in the process by which 5-MTP alleviates ER stress-mediated apoptosis. The results of the WB analysis revealed considerably higher expression levels of ATF6 and higher phosphorylation of PERK and IRE1α in the Nrf2KO+I/R+5-MTP group compared to WT controls (Figures 6A–D). Concurrently, proteins associated with ERS-mediated apoptosis (ATF4, CHOP, and DR5) and the phosphorylation of eIF2α increased significantly in Nrf2-KO mice (Figures 6E–H). To validate these findings, we performed IF staining to evaluate CHOP protein levels (Figures 6I,K). The Nrf2KO+I/R+5-MTP group presented higher CHOP expression than the WT+I/R+5-MTP group. Additionally, renal tissue apoptosis was evaluated via TUNEL staining (Figures 6J,L). The Nrf2KO+I/R+5-MTP group presented a higher number of TUNEL-positive cells. These findings suggest that 5-MTP mitigates ERS-mediated apoptosis through the activation of the Nrf2/HO-1 pathway.