Animal care and model construction

As previously described (Zhang et al. 2019), male C57BL/6 mice, aged 6-8 weeks, were purchased from the Experimental Animal Center of the First Affiliated Hospital of Wenzhou Medical University. The mice were housed under conditions of 22-25°C temperature, 40-60% humidity, and a 12-hour light/dark cycle, provided with standard chow and water. The mice were divided into four groups: sham (UUO7d and UUO14d) and unilateral ureteral obstruction (UUO) groups (UUO7d and UUO14d), with 6 mice per group. Under anesthesia with isoflurane, mice in the UUO groups underwent a midline incision to expose and ligate the left ureter, inducing obstruction, followed by suturing of the incision. The sham group underwent exposure of the left kidney without ureteral ligation. Mice were euthanized with isoflurane anesthesia at 7 and 14 days post-surgery, and kidneys were harvested for histological analysis. For the ischemia-reperfusion injury (IRI) model, an equal number of mice were randomly allocated to four groups: sham (IRI24h and IRI72h) and IRI groups (IRI24h and IRI72h). After administering anesthesia with isoflurane, two small incisions were made on the dorsal side to expose the kidneys. A non-traumatic microvascular clamp was used to occlude both renal pedicles for 40 minutes, blocking blood flow to the kidneys.

Following the ischemic period, the clamps were removed. Mice were sacrificed at 24 hours and 72 hours after reperfusion, and the kidneys were harvested for further analysis (Chen et al. 2023a). For the study of Aristolochic Acid Nephropathy (AAN), mice were randomly divided into two groups: the control group and the AAN group. Mice in the AAN group were administered Aristolochic Acid (Sigma-Aldrich, 3 mg/kg dissolved in PBS) via intraperitoneal injection every other day for 3 weeks, followed by a 3-week recovery period. Mice injected with PBS served as the control group. All mice were euthanized with isoflurane after 6 weeks of injections.

Clinical specimen collection

A total of 80 renal pathology slides from patients with IgA nephropathy, diabetic nephropathy, hypertensive nephropathy and lupus nephropathy in the First Hospital of Wenzhou Medical University were collected for the study, and they were classified into low-fibrosis (n=10) and high-fibrosis (n=10) groups according to the degree of fibrosis.

Histopathological examination

The renal specimens were preserved using 4% paraformaldehyde, embedded in paraffin, and sliced into 4 μm thick sections. According to the manufacturer's instructions, histological assessment of renal injury and fibrosis was performed on paraffin sections of mouse kidney tissue using Hematoxylin and Eosin (HE) staining (Solarbio, China) and Masson's trichrome staining (Solarbio, China). Twenty non-overlapping regions were randomly selected and imaged for each mouse. The severity of renal injury was assessed based on the intensity of tubular dilation (<25%, 25%-50%, 50%-75%, and 75%-100%, scored as 1, 2, 3, and 4, respectively). ImageJ software was utilized for quantitative analysis of renal fibrosis, expressed as the average percentage of fibrotic area relative to the total area.

Immunohistochemistry (IHC) staining

The kidney tissues were initially treated with xylene to remove wax and then progressively rehydrated through ethanol solutions (100%, 95%, 85%, and 75%), concluding with distilled water, followed by treatment with 3% hydrogen peroxide. Subsequently, the sections were blocked with goat serum for one hour, after which they were incubated with primary antibodies for 12-16 hours at 4°C, including anti-E-cadherin (Abcam, USA), N-cadherin (Abcam), type I collagen (Proteintech, USA), Vimentin (Proteintech), α-SMA (Santa Cruz, USA), and S100A2 (Abcam). Following PBS washing, HRP-labeled secondary antibodies were applied, and DAB chromogenic staining was performed, followed by hematoxylin counterstaining. Imaging was carried out using a Leica microscope.

Cell culture and treatment

Human kidney proximal tubular cells (HK-2), obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), were maintained in DMEM/DF-12 medium (Gibco, USA) enriched with 10% fetal bovine serum and 1% penicillin-streptomycin. The HK-2 cells were treated with TGF-β1 (20 ng/mL) for 48 hours to induce fibrosis-like changes. The cells were also exposed to 200 ng/ml of recombinant S100A2 protein (MCE, USA) or 50 μM SRT1460 (MCE) for 24 hours.

In this study, HK2 cells were treated with TGFβ1-IN-1 (HY-151427, MCE), a specific TGF-β1 inhibitor, at a concentration of 20 μM for 24 hours to evaluate its efficacy in blocking the effects of TGF-β1 on S100A2 expression. Additionally, to simulate hypoxia-induced renal injury, HK2 cells were exposed to 500 μM CoCl2 for 24 hours, and the subsequent changes in S100A2 expression were assessed before and after treatment.

Cell transfection

S100A2-siRNA was sourced from GenePharma (China). Prior to transfection, the cell culture medium was substituted with Opti-MEM (Invitrogen). Upon reaching approximately 70% cell density, transfection was conducted utilizing Lipofectamine 2000 (Invitrogen), following the manufacturer's protocol. The siRNA (50 μM) and Lipofectamine 2000 (5 μl) were separately mixed with Opti-MEM (250 μl). After a 15-minute incubation period, the diluted solutions of siRNA (250 μl) and Lipofectamine 2000 (250 μl) were combined and introduced to HK-2 cells for incubation. Following 24 hours, the medium was replaced with 10% FBS medium. Subsequently, TGF-β1 (20 ng/mL) was introduced to the HK-2 cells and allowed to incubate for an additional 24 or 48 hours prior to RNA or protein extraction.

RT-qPCR

Total RNA was isolated from kidney tissues and HK-2 cells using Trizol reagent (Glpbio, USA). Subsequently, the RNA was reverse transcribed to generate cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, China). Quantitative PCR experiments were conducted using SYBR Green reagent (Vazyme), with β-actin serving as an endogenous reference gene. For quantitative analysis, all samples were assessed using the 2-ΔΔCt method. The relevant primer sequences are provided in Supplementary Table 1.

Immunoblotting and Co-inmunoprecipitation (Co-IP) experiments

Kidney tissue and HK-2 cell proteins were isolated and separated using SDS-PAGE gels and subsequently transferred onto a PVDF membrane. After blocking with 5% non-fat milk, the membrane was probed with the relevant primary antibodies overnight including anti-Vimentin (Santa Cruz), E-cadherin (Abcam), α-SMA (Santa Cruz), N-cadherin (Abcam), COL1 (Proteintech), S100A2 (Abcam), Smad2/3 (Abcam), p-Smad2/3 (Thr8, Abcam), p-PI3K (Tyr607, Affinity, USA), PI3K (Proteintech), p-AKT (Ser473, CST), AKT (CST), Sirt1 (Affinity), p-FoxO1 (Ser256, Affinity), FoxO1 (Affinity), Snail1 (Abcam), COL3 (Proteintech), GAPDH (Proteintech), H3 (Proteintech), and β-actin (Affinity). On the following day, the membrane was washed and incubated with the corresponding HRP-labeled secondary antibody. Chemiluminescence was then developed, and the band intensity was quantified with ImageJ software. For Co-IP experiments, the protein supernatant, lysed using Western and IP cell lysate (Beyotime, China) with 1% PMSF, underwent pre-incubation with Protein A+G agarose beads at 4°C for 30 minutes to minimize non-specific binding. Subsequently, the mixture was evenly distributed into four 1.5 mL tubes. Samples from three tubes were then subjected to overnight incubation with magnetic beads pre-bound with rabbit IgG (Beyotime), anti-S100A2 (Abcam), and anti-FoxO1 (Affinity) on a shaker at 4°C, while the remaining tube was left untreated. Following washes with TBS (Beyotime), the proteins were dissolved in loading buffer and subsequently heated at 95°C for 5 minutes for subsequent immunoblotting analysis.

Transcriptome sequencing and bioinformatics analysis

After transfection, HK-2 cells were collected and suspended in 1 mL of Trizol reagent. The samples were then sent to Personalbio (Shanghai, China) for transcriptome sequencing. The resulting data were analyzed and visualized using the R software packages "clusterProfiler," "DOSE," "org.Hs.eg.db," "stringr," and "ggplot2."

Immunofluorescence staining

HK-2 cells were first fixed onto slides, followed by washing and permeabilization with 0.1% Triton X100, and blocking with goat serum. Then cells were placed at 4°C and exposed to primary antibodies overnight, including anti-E-cadherin (Abcam), anti-type I collagen (Proteintech), anti-type III collagen (Proteintech), anti-N-cadherin (Abcam), S100A2 (Abcam), FoxO1 (Affinity), p-FoxO1 (Ser256, Affinity), anti-α-SMA (Santa Cruz), and anti-p-Smad2/3 (Thr8, Abcam). Subsequent incubation with secondary antibodies conjugated with Alexa Fluor 488 or 588 (Invitrogen, USA) followed. Finally, DAPI was used for nuclei visualization, and microscopy (Leica) was utilized for image capture.

EMT detection

To evaluate epithelial-to-mesenchymal transition (EMT), HK2 cells were cultured under standard conditions using DMEM/DF12 medium and treated with TGF-β1 to induce EMT. Additionally, a mouse model of renal fibrosis was established. Morphological changes in the cells were assessed using optical microscopy. EMT markers such as E-cadherin, N-cadherin, vimentin, and α-SMA, were analyzed at both cellular and tissue levels using Western blotting with specific antibodies. Immunofluorescence staining was employed to examine the localization and expression of EMT markers, while immunohistochemistry (IHC) was utilized to determine the expression of these markers in tissue samples. RNA was extracted and reverse transcribed into cDNA, followed by quantitative PCR to quantify the mRNA levels of EMT-related markers. This comprehensive approach allowed for detailed assessment of EMT at various biological levels.

Statistical analysis

Data were analyzed statistically using GraphPad Prism 9.0. The comparison between two groups was conducted using the t-test, while one-way analysis of variance (ANOVA) was employed for comparisons among multiple groups. Statistical significance was considered when P < 0.05.