Human conditionally immortalized glomerular podocytes (HPCs) were cultured in complete RPMI 1640 medium containing 10% fetal bovine serum (Gibco, Gaithersburg, MD, United States) and insulin-transferrin-selenium (1×, Thermo Fisher Scientific, Waltham, MA, United States) at 33°C in 5% CO₂. After differentiation at 37°C for 10–14 days, the mature podocytes were randomly divided into three groups: Control, PAN-stimulated group (PAN, 50 μg/mL, GLPBIO), and TP intervention group (5 nM, Yuanye Bio-Technology Co., Ltd, Shanghai). Additionally, 5′-azacytidine (5-AzaC, 10 μM in phosphate-buffered saline [PBS], MCE, United States) or RG108 (Beyotime, Shanghai, China) were used as the DNA methyltransferase inhibitors. Lentivirus (Lv) carrying the TET2 short-hairpin RNA (shRNA) and control lentivirus (scramble Lv) were purchased from GeneChem (Shanghai, China). After incubation for 24–72 h, cells were collected for subsequent experiments.

HPCs were treated with PAN and TP for 24 h, then incubated with 10 μM DCFH-DA (Beyotime, Shanghai, China) for 30 min, followed by washing three times with PBS. Thereafter, the fluorescence intensity of DCFH-DA was detected using fluorescent microscopy.

The permeability assay was conducted as previously described [15]. Briefly, HPCs (1 × 10⁵/well/200 μL) were seeded in the upper layer of the transwell chamber (24 well plate, NEST Biotechnology Co. Ltd, Wuxi, China). Cells were treated with PAN (50 μg/mL) or TP (5 nM) after undergoing starvation. After 36 h, fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (0.25 mg/mL, Solarbio, Beijing, China) was added to the upper chamber of the transwell chamber for 10 min at room temperature. The fluorescence intensity of the medium in the lower chamber, reflecting the podocyte monolayer permeability, was detected using a multifunctional enzyme-linked immunosorbent assay reader (excitation: 493 nm, emission: 550 nm; Thermo Fisher Scientific, Waltham, MA, United States).

Total RNA was extracted from HPCs using the TRIzol reagent and reverse-transcribed to cDNA using a Primescript™ reagent kit (TaKaRa, Kusatsu, Shiga, Japan). The primers used were as follows: ZO-1-Forward: 5′-CAA​CAT​ACA​GTG​ACG​CTT​CAC​A′, ZO-1-Reverse: 5′-CAC​TAT​TGA​CGT​TTC​CCC​ACT​C-3′; TET2-Forward: 5′-TCC​TGG​TGG​CAG​CTC​TGA​ACG-3′, TET2-Reverse: 5′-TGG​TGG​TGG​TGG​TGT​GGT​AGT​G-3’; GAPDH-Forward: 5′-ACC​ACA​GTC​CAT​GCC​ATC​AC-3′, GAPDH-Reverse: 5′-TCC​ACC​ACC​CTG​TTG​CTG​TA-3′. PCR was performed using a QuantStudio 5 real-time PCR system (Applied Biosystems, Waltham, MA, United States). GAPDH was used as an internal reference. Each experiment was performed in triplicate.

HPCs were lysed using RIPA buffer containing a proteinase inhibitor cocktail at 4°C for 30 min and centrifuged at 10,000 × g for 10 min. The BCA reagent kit (Beyotime, Shanghai, China) was used to calculate the protein concentration in the supernatant, followed by mixing with SDS-PAGE sample loading buffer (2×) and boiling at 95°C for 10 min. Equal amounts of protein were separated using SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% skim milk, and then incubated with primary antibodies, including TET2 (Abcam, Cambridge, UK, ab94580), ZO-1 (ABclonal, Wuhan, China, A0659), NOX4 (ProteinTech, Rosemont, IL, United States, 67,681-1-1g), and GAPDH (ProteinTech, Rosemont, IL, United States, 60,004-1-Ig) at 4°C overnight, followed by incubation with infrared-labeled anti-rabbit/mouse IgG antibody. Reactive bands were observed using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, United States).

Podocytes were fixed with 10% formalin for 10 min at room temperature, blocked with 5% goat serum, and incubated with rabbit anti-ZO-1 (ABclonal, Wuhan, China) at 4°C for 1 h. Podocytes were then incubated with FITC-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Waltham, MA, United States) at 4°C for 1 h after washing with PBS three times. Cells were photographed under a fluorescence microscope (Olympus) after staining with DAPI.

DNA was isolated using the genome extraction kit (Genefist, Shanghai, China), according to the manufacturer’s protocol. The genome was broken into 200–1000 bp segments by ultrasonic fragmentation. The ultrasonic conditions were set as: sonicate 3-4 pulses of 10 s each at 50 W, followed by 30 s rest on ice between each pulse. The sonicated cell lysate was analyzed by agarose gel analysis before subsequent use.

Methylated DNA was isolated from sonicated DNA (1.0 μg) using the EpiQuik™ Methylated DNA Immunoprecipitation Kit (Epigentek, Farmingdale, NY, United States), according to the manufacturer’s protocol. The EpiQuik™ Hydroxymethylated DNA Immunoprecipitation Kit (Epigentek, Farmingdale, NY, United States) was used to extract hydroxymethylated DNA from the sonicated DNA (0.5 μg) in each group. Meanwhile, total sonicated DNA was used as the input DNA for equal loading.

For DNA amplification, the PCR reactions were performed with a volume of 20 μL containing: TaKaRa Ex Taq^® (2×) 10 μL, forward primer (10 μM) 1 μL, reverse primer (10 μM) 1 μL, eluted DNA 2 μL, and ddH₂O 6 μL. The primer sequences were as follows: Pro-ZO-1-Forward: 5′-AAC​GAG​AGC​AAC​GCT​TCT​GA-3′ and Pro-ZO-1-Reverse: 5′-GTC​CAC​TTG​CTC​CTC​GAC​AA-3′. All reactions were performed on a Veriti^® 96-Well Thermal Cycle (Thermo Fisher Scientific), and the PCR products were observed using a Gel Imaging System.

Statistical analyses were performed using SPSS (version 20.0, IBM Corp., Armonk, NY, United States). Values are presented as mean ± standard deviation. Quantitative data were compared between the two groups using the student’s t-test. One-way analysis of variance was used for multiple group comparisons. p-value <0.05 was considered statistically significant.

PAN induces podocyte injury by causing decreased expression and abnormal distribution of SD proteins, including ZO-1 [16]. Similarly, in our study, we observed a decrease in the fluorescence intensity of ZO-1, along with its discontinuous distribution in PAN-stimulated podocytes (Figures 1A, B). ZO-1 mRNA and protein expression levels were also decreased (Figures 1C–E). However, TP treatment markedly increased the fluorescence intensity and upregulated ZO-1 expression levels. Next, we assessed podocyte permeability using a transwell chamber, and found that the permeability of PAN-treated podocytes was nearly twice of that of untreated podocytes (Figure 1F). However, TP intervention significantly decreased the podocyte permeability of the PAN-treated group. These findings suggest that TP can decrease podocyte permeability by upregulating ZO-1 expression.

Many studies have shown that PAN-induced podocyte injury is associated with the overproduction of ROS [8]. Considering that podocytes express various NADPH oxidase subunits, including NOX4, we determined the cellular NOX4 and ROS levels. The results showed that PAN significantly increased the cellular ROS level and NOX4 expression (Figures 2A, C). However, TP significantly reduced the ROS level and NOX4 expression, which was confirmed by quantitative analysis (Figures 2B, D). Apocynin, an inhibitor of NADPH oxidase activity, led to a significant reduction in the ROS level induced by PAN. These results correlate with the changes in podocyte permeability, and indicate that PAN-induced oxidative stress might be involved in the increased podocyte permeability.

Growing evidence has shown that epigenetics played an important role in the evolvement of podocyte injury. Therefore, we detected the expression of DNA demethylase TET family proteins. qRT-PCR and western blotting results indicated that the expression of TET2 was significantly reduced in the PAN group compared with the control group (Figure 3). After TP treatment, the expression of TET2 increased significantly (Figure 3). However, TET2 and TET3 demonstrated no significant differences among the groups (data not shown). These results indicate that TP may selectively regulate the expression of TET2 in PAN-induced podocyte injury model.

It is well known that the TET family proteins play a crucial role in passive and active demethylation [17]. The synchronous upregulation of TET2 and ZO-1 suggests that ZO-1 expression may be regulated by TET2. To test our hypothesis, we firstly obtained the promoter region of ZO-1 by retrieving the upstream 2,200 bp sequence of the transcription start site from the human genome. Using the online methylation analysis software MethPrimer ¹ , we found that the ZO-1 promoter region contains three CpG islands (688–858 bp, 1,614–1,874 bp, and 1,950–2,145 bp; Criteria used: Island size >100, GC Percent >50.0, Obs/Exp >0.6), indicating that the expression of ZO-1 may be epigenetically regulated (Figure 4). Subsequently, we used the DNA methyltransferase inhibitors 5-azacytidine (5-AzaC) and RG108 to treat the podocytes. The results showed that ZO-1 expression was significantly increased in podocytes treated with 5-AzaC and RG108 compared with the PAN alone stimulated group (Figure 5). Taken together, these results suggest ZO-1 expression is closely related to epigenetic regulation.

To further test whether TP regulates the methylation status of the ZO-1 promoter, we used MeDIP and hMeDIP methods to analyze the methylation status of ZO-1 in genomic DNA samples isolated from podocytes. As expected, the decreased expression of ZO-1 in the PAN group was associated with increased promoter methylation, while the promoter methylation levels of ZO-1 in the TP treatment group were significantly reduced (Figure 6A). Furthermore, we observed that ZO-1 demethylation upon TP intervention corresponded with increased ZO-1 hydroxymethylation (Figure 6B), suggesting that TP can mediate ZO-1 demethylation, which is accompanied by ZO-1 hydroxymethylation.

In order to determine the role of TET2 in ZO-1 expression and methylation, we knocked down TET2 using a lentivirus-mediated shRNA prior to TP intervention. After 72 h of infection, most podocytes strongly expressed GFP, indicating strong infectivity of the Lv (Figure 7A). qRT-PCR results showed the mRNA expression of TET2 in the TET2 Lv treatment group decreased by approximately 67.57% compared to that in the control group (Figure 7B). Western blot results also revealed that TET2 was significantly inhibited after Lv treatment (Figures 7C, D). The podocyte permeability increased significantly in the TET2 knockdown group compared with the scrambled Lv group (Figure 7E). Moreover, the expression of ZO-1 was decreased significantly in the TET2 knockdown group compared with the scrambled Lv group, even after treatment with TP (Figures 7F, G). Meanwhile, compared with the control group, increased promoter methylation of ZO-1 was observed in the TET2 knockdown group (Figure 8A), accompanied by decreased ZO-1 hydroxymethylation (Figure 8B). Altogether, these results indicate TP may upregulate the expression of ZO-1 through TET2-mediated 5 mC demethylation.