Mefunidone (Lot No. 21062601) was synthesized by the Pharmaceutical Sciences Department, Central South University (Changsha, China). Folic acid (FA) (F8758) and sodium bicarbonate (V900182) were purchased from Sigma-Aldrich. Antibodies against proteins NGAL (ab216462) and collagen Ⅰ (ab270993) were purchased from Abcam. Antibodies against Bax (^#2772), cleaved-caspase3 (^#9664), and α-smooth muscle actin (α-SMA) (^#19245) were purchased from Cell Signaling Technology. Antibodies against proteins bcl2 (^#T40056) were purchased from Abcam. Antibodies against OxPhos (45–8,099) were purchased from Invitrogen. Antibodies against E-cadherin (20874-1-AP), vimentin (60330-1-Ig), α-tubulin (66031-1-Ig), and GAPDH (^#60004-1-Ig) were purchased from Proteintech. A tissue reactive oxygen species assay kit (DHE) (^#D7008) was purchased from Sigma-Aldrich. FITC Annexin V and PI staining kit (556547) was purchased from BD Pharmingen. Cell counting kit-8 (K1018) was purchased from APExBIO Technology LLC. The TMRM assay kit (T668) was purchased from Thermo Fisher Scientific. Blood urea nitrogen (BUN) (C013-2-1) and serum creatinine (SCr) (C011-2-1) kits were obtained from the Nanjing Jiancheng Bioengineering Institute. All the chemicals used in this study were of analytical grade.

Male C57BL/6 mice, weighing 20–22 g (6–8 weeks old), were obtained from Silaike Laboratory (Shanghai, China). All animals were housed in a pathogen-free environment with a light/dark cycle of 12 h. All animals were fed an ad libitum diet and water, with adaptive feeding for at least 7 days. All animal experiments were conducted strictly in accordance with the standards of humane treatment established by the Laboratory Animal Sciences Association and Laboratory Animal Sciences Center at Central South University.

Human proximal renal tubular epithelial cells (HK-2) were purchased from Procell Life Science&Technology Co., Ltd. (Wuhan, China) and cultured in DME/F12 medium (Gibco, United States) supplemented with 1% penicillin-streptomycin (Gibco, United States) and 10% fetal bovine serum (FBS, Gibco, United States) under 5% CO₂ and 95% air atmosphere at 37°C. HK-2 cells were cultured in a medium containing CoCl2 (7791-13-1, Changsha Jingkang New Material Technology Co. LTD., China) without nutrients (serum-free, glucose-free) for 6 h. The medium was then replaced with DME/F12 with or without mefunidone for 12 h to induce the hypoxia/reoxygenation (H/R) model.

Serum creatinine and blood urea nitrogen were detected according to the kit instructions (Nanjing Jiancheng Bioengineering Institute).

Paraffin-embedded kidney tissue sections (4 µm-thick) were stained with hematoxylin-eosin (HE) and/or Masson’s trichrome for pathological analysis. The percentage of cortical tubules was semi-quantitatively graded in a blinded manner, as reported previously (Jin et al., 2013) (Jiang et al., 2019). Collagen accumulation was measured using Masson’s trichrome staining, with blue collagen deposition as a positive signal. The percentage of positive areas in the entire field of vision (except for large vessels) was sorted by 0–4 points: 0, normal; 1, <25%; 2, 25%–50%; 3, 50%–75%; and 4, >75% (Lin et al., 2005; Fang et al., 2019).

The sliced kidney sections were dewaxed, rehydrated, and treated with H₂O₂ (3%) for 20 min, pepsin (0.4%) for 20 min, and then incubated with 5% bovine serum albumin in PBS for 30 min. The sections were incubated overnight with anti-NGAL, anti-collagen I, or anti-vimentin antibodies at 4°C. The samples were then washed with PBS and incubated with secondary antibodies (Abcam, Cambridge, United Kingdom).

Dihydroethidium (DHE) staining was performed to detect the ROS levels. Cryosections of the kidney tissue (4 µm) were incubated in the dark for 30 min at 37°C for staining. A fluorescence microscope (Nikon, Tokyo, Japan) was used to test ethidium fluorescence with excitation/emission at 488/610 nm.

Fixed kidney tissues (1 mm) were incubated overnight at 4°C with 2.5% glutaraldehyde, followed by chemical treatment with osmium tetroxide. The kidney tissues were subsequently rinsed, fixed, paraffin-embedded, and mitochondrial morphology was observed and recorded using transmission electron microscopy (TEM).

RNA was extracted from the kidney tissues using TRIzol reagent (Invitrogen). cDNA was synthesized from total RNA using the Real Master Mix (Bio-Rad Laboratories, CA, United States). mRNA expression was detected using SYBR Green assay (Takara Bio Inc., Kusatsu, Japan). β-actin was used to normalize the mRNA ratios, and the mean ± standard error of the mean (SEM) was used to represent the final data. The primer sequences used were as follows: mouse MnSOD: forward, 5′-CAG​ACC​TGC​CTT​ACG​ACT​ATG​G-3′; reverse, 5′-CTCGGTGGCGTTGAG ATTGTT-3′; mouse CAT: forward, 5′-CCT​ATT​GCC​GTT​CGA​TTC​TC-3′; reverse, 5′-CCC​ACA​AGA​TCC​CAG​TTA​CC-3′; and mouse β-actin: forward, 5′-CTGTCCCTG TATGCCTCTG-3′; reverse, 5′-TTG​ATG​TCA​CGC​ACG​ATT-3′.

Proteins from mouse kidney tissues were extracted in ice-cold 2 × SDS, and the protein concentration was detected using a BCA protein kit (#23225, Thermo, United States). Proteins were separated by 8%–15% SDS-PAGE and transferred to PVDF membranes (Millipore, MA, United States). The membranes were blocked in 5% skim milk prepare by TBST (150 mmol/L NaCl, pH 7.6, 0.05% Tween 20, and 20 mmol/L Tris-HCl) for 1 h at room temperature. The membranes were incubated with the primary antibody at 4 C overnight, followed by hybridization with HRP-conjugated secondary antibodies at room temperature for 1 h the next day. ECL western blotting reagents (32106, Thermo, United States) were used to visualize the results, and ImageJ was used for quantification.

An in situ cell death detection kit (11684795910, Roche, Switzerland) was used to evaluate the kidney tissue sections of 4 µm-thick treated with paraffin embedding, following the manufacturer’s protocols.

Effect of mefunidone on cell viability by Cell Counting Kit-8 (CCK-8). Briefly, HK-2 cells were seeded at a density of 4 × 10³ cells/well in 96-well plates. After overnight culture, the cells were incubated with different doses of mefunidone (0, 20, 40, 80, 120, 160 μg/ml) in 96-well plates for 24 h. HK-2 cells were then incubated with CCK-8 solution at 37°C for 2 h, and cell viability was determined by measuring the absorbance at 450 nm with a microplate reader.

HK-2 cells were seeded in 6-well plates and divided into three groups: control, H/R, and H/R plus mefunidone. Hypoxia injury was induced by a medium containing CoCl₂ without nutrients (serum-free, glucose-free) for 6 h; then, the medium was replaced with DME/F12 with or without mefunidone (80 μg/ml) for 12 h. HK-2 cells were collected in centrifuge tubes, according to the manufacturer’s instructions, and Annexin V and PI staining kit was used to assess cell apoptosis by flow cytometry.

HK-2 cells were seeded in 6-well plates and divided into three groups: control, H/R, and H/R plus mefunidone. Hypoxic injury was induced using a medium containing CoCl₂ without nutrients (serum-free, glucose-free). After 6 h, the medium was replaced by DME/F12 with or without mefunidone (80 μg/ml) for 12 h. Collecting HK-2 cells in centrifuge tubes, after adding TMRM reagent, mitochondrial membrane potential was monitored by flow cytometry.

All data are expressed as mean ± standard error of the mean (SEM). One-way ANOVA with post hoc analyses using the LSD method was used for comparisons among groups. Statistical significance was set at p < 0.05.

The effect of mefunidone on IRI-induced AKI was determined by HE staining of kidney tissue sections (Figures 1A,B). Compared to the sham group, the IRI-treated group showed obvious kidney tubular injury, including proximal tubular brush border loss, tubular lysis, and cast formation. Mefunidone partially protected the tubular epithelium from proximal tubular brush border loss, tubular lysis, and cast formation, with remarkable effects at a concentration of 100 mg/kg/day. Compared with the sham group, the IRI-treated group showed significant renal dysfunction, and the mefunidone treatment group showed partially improved renal function (Figures 1C,D). These results indicated that different doses of mefunidone could attenuate renal IRI to varying degrees, and the most obvious effect was observed at 100 mg/kg/day. Therefore, a dose of 100 mg/kg/day was selected for all the subsequent experiments. These results were also supported by the decreased expression of NGAL observed using IHC and western blotting in the mefunidone-treated group (Figures 1E–G).

In this study, we explored the effects of long-term ischemia on fibrosis in IRI. We assessed the effects of mefunidone on day 14 after IRI modeling. Compared with the IRI-treated group, treatment with mefunidone significantly improved renal tubular injury and renal fibrosis in CKD induced by IRI, as shown in Supplementary Figure S1.

Consistent with renal tubular damage, ROS accumulation was observed in the kidneys after IRI injury, and mefunidone treatment noticeably reduced ROS accumulation (Figure 2A). Mefunidone treatment also significantly decreased IRI-induced levels of serum MDA, a marker of lipid peroxidation (Chen et al., 2019a; Li et al., 2019a) (Figure 2B). In addition, the mRNA expression of catalase (CAT) and manganese superoxide dismutase (mnSOD) was elevated in the IRI-treated group, and treatment with mefunidone reversed these trends (Figures 2C,D).

Previous studies have shown that apoptosis of renal tubular epithelial cells is involved in the pathological process of renal injury induced by IRI (Priante et al., 2019; Ma et al., 2020; Pan et al., 2020). TUNEL staining was performed to investigate whether mefunidone can improve IRI-mediated renal apoptosis. The results revealed that mefunidone treatment significantly attenuated renal apoptosis following IRI-induced AKI (Figures 3A,B). Subsequently, we examined the expression of cleaved-caspase3, which is an apoptotic marker. We observed an increase in cleaved-caspase3 expression in the IRI-treated group, whereas mefunidone treatment decreased the expression of cleaved-caspase3 (Figure 3C). Then we verified the therapeutic effect of mefunidone in vitro. First, we used CCK-8 to determine the optimal drug concentration of mefunidone for 24 h for HK-2 cell viability. As shown in Supplementary Figure S2, 80 μg/ml mefunidone showed no obvious toxicity to HK-2 cells. We selected a concentration of 80 μg/ml mefunidone for the following experiments. We used flow cytometry to detect apoptosis of HK-2 cells, consistent with animal experiments. The apoptosis rate of HK-2 cells in the H/R group was relatively high, and the number of apoptotic HK-2 cells decreased significantly after treatment with mefunidone (Figures 3D,E). We also detected the cleaved-caspase3 expression in the H/R model of HK-2 cells. The cleaved-caspase3 expression was increased in the H/R group, and mefunidone downregulated the expression of cleaved-caspase3 (Figure 3F).

We used TEM to detect the mitochondria, and the results clearly showed the mitochondrial cristae and intact mitochondrial membrane in the sham group. Mitochondrial swelling and vacuolation appeared in the IRI-treated group, although the mitochondrial integrity status remained unchanged, and mitochondrial damage partially improved after mefunidone treatment (Figure 4A). Since we observed that mefunidone could improve mitochondrial damage and that the mitochondrial pathway is the most important apoptotic mechanism in AKI, we examined whether mefunidone treatment abated IRI-mediated renal apoptosis by inhibiting the expression of mitochondrial-related pro-apoptotic proteins. Western blot analysis confirmed that in the IRI-treated group, Bax expression increased while bcl2 expression decreased. Mefunidone treatment reduced Bax expression and increased bcl2 expression (Figures 4B,C). Moreover, we evaluated mitochondrial electron transport chain protein expression and found that complex I, complex II, complex III, and complex V protein expression in the IRI-treated group decreased significantly compared with those in the sham group, and mefunidone partially recovered complex III and complex V, but not the downregulation of complex I and complex II (Figures 4D,E).

To verify the protective effect of mefunidone on mitochondria in vitro, we measured the expression of mitochondria-related apoptotic proteins (Figures 5A,B). We found that mefunidone downregulated the expression of the pro-apoptosis-related protein Bax while upregulating bcl2 expression, which is in accordance with our observations in vivo. Subsequently, we detected changes in MMP and the mitochondrial electron transport chain complex expression in HK-2 cells. As shown in Figure 5C, mefunidone treatment significantly improved the MMP decline caused by the H/R model. Compared with the control group, complex I, complex II, complex III, and complex V protein expression decreased significantly in the H/R group, and mefunidone partially recovered complex III and complex V but did not downregulate complex I and complex II (Figures 5D,E).

To explore whether mefunidone also protects against FA-induced AKI, HE staining was applied to observe and evaluate the severity of tubular injury induced by FA in AKI. Compared to the sham group, the model group receiving FA injection showed obvious tubular dilatation, brush border deletion, cell debris, and vacuolization. The above situation significantly improved after mefunidone treatment (Figures 6A,B). Analysis of renal function revealed that serum creatinine and blood urea nitrogen in FA treated group were significantly higher in the FA-treated group than in the sham group. The mefunidone treatment group partially reduced the serum creatinine and blood urea nitrogen levels to maintain renal function (Figures 6C,D). These results were also supported by the decreased expression of NGAL in the mefunidone-treated group, as determined by western blotting (Figure 6E). Similarly, to determine whether mefunidone could also inhibit apoptosis in the FA-induced AKI model, we examined the expression of the apoptosis marker cleaved-caspase3. We observed an increase in cleaved-caspase3 expression in the FA group, whereas mefunidone treatment decreased the expression of cleaved-caspase3 (Figure 6F).