The human renal proximal tubular HK-2 cell line (Cat. No. CL-0109) was acquired from Procell Life Science & Technology Co., Ltd. (Wuhan, China). PA (C16:0) (Cat. No. P5585) and streptozotocin (Cat. No. S0130) were obtained from Sigma-Aldrich (St. Louis, MO, United States). Atorvastatin was purchased from The First Affiliated Hospital of Guangzhou University of Chinese Medicine. Anti-NLRP3 (Cat. No. 27458-1-AP), anti-CD36 (Cat. No. 18836-1-AP), anti-ASC (Cat. No. 10500-1-AP), anti-cleaved-caspase-1 (Cat. No. 81482-1-RR), anti-cleaved-IL-1β (Cat. No. AF4006), Anti-Cleaved-GSDMD (Cat. No. 36425), Anti-GSDMD (Cat. No. 39757), Anti-KIM-1 (Cat. No. ab78494), Anti-L-FABP (Cat. No. 13626-1-AP), Anti-NGAL (Cat. No. 26991-1-AP), and anti-α-tubulin (Cat. No. 11224-1-AP) were purchased from Proteintech Group, Inc. (Wuhan, China). RIPA lysis buffer (Cat. No. P0013D), the BCA kit (Cat. No. 010719190225), and the SDS-PAGE gel preparation kit (Cat. No. 120120201209) were purchased from Beyotime Biotechnology (Shanghai, China). The Oil Red O staining kit (Cat. No. G1262) was obtained from Solarbio Science & Technology (Beijing, China). AS-IV (purity > 98% as determined by HPLC) was purchased from Nanjing Chunqiu Bioengineering Co., Ltd. (Nanjing, China).

Male Sprague-Dawley rats (170 ± 10 g) were acquired from the Experimental Animal Center at Guangzhou University of Chinese Medicine. After 1 week of adaptive feeding, the rats were randomly divided into two groups: the control group (normal diet, n = 6) and the high-fat diet (HFD) group (n = 30). The experimental method refers to our previous study (Li et al., 2023). Rats were considered diabetic after three consecutive days of fasting blood glucose levels exceeding 16.7 mmol/L. Rats in the HFD group were randomly assigned to one of five groups (n = 6 rats per group): the DKD model group, the positive control group receiving atorvastatin treatment (10 mg/kg, administered via gavage), and the AS-IV treatment groups (20, 40, and 80 mg/kg/day). Throughout the experiment, body weight was evaluated on a weekly basis. Additionally, fasting blood glucose (FBG) was tested every 4 weeks after a 6-h period of fasting. 24-h urine samples were collected using individual metabolic cages. All rats were sedated and sacrificed after 12 weeks of treatment. Serum was collected for biochemical analysis. For pathological analysis, the left kidneys were immersed in paraffin, while the right kidneys were preserved at −80°C. Approval for this study was granted by the Animal Ethics Committee of the First Affiliated Hospital of Guangzhou University of Chinese Medicine (approval number TCMF1-2019009).

The rat microalbuminuria ELISA Kit (Cat. No. CSB-E12991r) was utilized to determine the urinary albumin concentration. The urinary creatinine level was measured using the Creatinine Assay Kit (Cat. No. C011-2-1, Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s instructions. The levels of serum triacylglycerol (TG), total cholesterol (TC), low-density lipoprotein–cholesterol (LDL-C), blood urea nitrogen (BUN), and serum creatinine (Scr) were measured using a Roche automatic biochemistry analyzer (Model No. Cobas c702, Roche, Japan) at the Laboratory Department of the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine.

To evaluate the histology of the kidneys, the kidney tissues were first treated with 4% paraformaldehyde for 48 h, followed by dehydration and embedding in paraffin. Thin tissue sections (4 μm) were prepared for staining with hematoxylin and eosin (H&E), periodic acid–Schiff (PAS), and Masson’s trichrome. To perform immunofluorescence staining, the kidney sections were treated with anti-NLRP3 as per the guidelines provided by the manufacturer. Staining analysis was conducted using light microscopy by two separate blinded observers. The collected images were assessed using ImageJ 1.8.0 (National Institutes of Health, United States).

HK-2 cells were cultured in DMEM/F12 medium (Cat. No. BL305A, biosharp, China) supplemented with 10% fetal bovine serum (Cat. No. 10100147, Gibco, United States), 100 μg/mL streptomycin, and 100 U/mL penicillin (Cat. No. 10100147, Gibco, United States) in a 5% CO₂ incubator at 37°C. Cells were divided into five groups: (1) the control group (cells treated with 1% BSA), (2) the PA group (cells treated with 400 mM PA), and (3–5) the PA + AS-IV groups (20, 40, and 80 µM AS-IV). To investigate the impact of AS-IV on CD36 expression in PA-induced HK-2 cells, we enhanced the expression of CD36 in HK-2 cells by utilizing a lentiviral expression vector. The CD36 overexpression adenovirus was acquired from Genechem Co., Ltd. (Shanghai, China). HK-2 cells in the 80 μM AS-IV group were transfected with the CD36 overexpression plasmid. Western blot analysis and PCR were used to determine the expression levels of CD36 in HK-2 cells.

We assessed the toxic effects of AS-IV by employing the Cell Counting Kit-8 (CCK-8, MIKX, Shenzhen, China). HK-2 cells were cultured in 96-well plates at a density of 1 × 10⁴ cells/mL in 100 μL of culture medium for 48 h. Different concentrations of AS-IV (0, 20, 40, 80, and 120 μM) were added, with or without PA (400 mM), in 10 μL. After incubating for 2 h, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated again. The absorbance at 450 nm was determined with a microplate reader (Thermo Fisher Scientific, Waltham, United States). The experiments were carried out in triplicate.

HK-2 cells were stained with Oil Red O according to the manufacturer’s instructions, following the experimental steps described in our previous study (Li et al., 2023). HK-2 cells were seeded into six-well dishes and incubated, and the initial medium was replaced. The cells were rinsed two times using PBS, treated with Oil Red fixative for 30 min, and finally washed two times with distilled water. Afterwards, the cells were rinsed with 60% isopropanol for 20–30 s, which was then removed; subsequently, Oil Red O staining solution was added. After 20 min, the Oil Red O solution was disposed of, and the cells were washed using 60% isopropanol for 20–30 s until the interstitial space turned transparent. The dishes were additionally rinsed five more times using distilled water until all excess stain was removed. Afterwards, Oil Red O buffer was introduced and removed within 1 min. In the end, purified water was included to ensure the cells were fully covered for examination using a fluorescence microscope.

Total RNA was extracted from both rat kidney tissues and HK-2 cells using TRIzol reagent (Ca. No. QP020, MRC, United States) and then converted to cDNA using the FastKing RT Kit (Ca. No. KR116, TIANGEN, China) following the manufacturer’s instructions. A CFX Connect Real-Time PCR System (Bio-Rad, United States) was utilized for quantitative RT-PCR analyses. Primer sequences are presented in Table 1.

Total protein was extracted from HK-2 cells or rat renal tissues using RIPA lysis buffer (Cat. No. P0013B, Beyotime, China). The protein concentration was determined using a BCA protein assay kit (Cat. No. P0010S, Beyotime, China) according to the provided instructions. The protein samples were mixed with 5× loading buffer (Cat. No. FD002, Fdbio science, China) and boiled for 5 min. Proteins were separated by SDS-PAGE and then transferred to membranes, which were incubated at room temperature in 5% skimmed milk for 1 h and subsequently exposed to primary antibodies (anti-CD36, anti-NLRP3, anti-cleaved-IL-1β, anti-cleaved-Caspase-1, anti-ASC, anti-GSDMD, anti-cleaved-GSDMD, anti-KIM-1, anti-L-FABP, anti-NGAL, and anti-α-tubulin) overnight at 4°C. Afterward, the membranes were washed, incubated with goat anti-rabbit for 1 h at ambient temperature, and washed again. Enhanced chemiluminescence (ECL, Ca. No. WBKLS0500, Millipore, United States) was performed using Clarity Max™ Western ECL Substrate (Ca. No.1705062, Bio-Rad, United States). The western blots were analyzed with ImageJ software (NIH, MD, United States).

HK-2 cells were seeded into six-well dishes, incubated, washed, trypsinized, suspended in PBS, incubated with MitoSox Red (Cat. No.M36008, Thermo Fisher Scientific Inc., United States) at 37°C for 30 min, washed three times with PBS, and centrifuged. The supernatant was discarded, and flow cytometry analysis was conducted using a FACSCalibur flow cytometer (BD, United States).

All data are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 9.5.1 software and SPSS 26.0 software. One-way analysis of variance (ANOVA) and Tukey’s multiple comparison test were used for comparisons among all groups. The t-test was used for pairwise comparisons. Differences were considered statistically significant if p < 0.05.

Initially, there were no obvious disparities among the six groups in body weight (Figure 1A). However, by week 12, the control group had a significantly higher body weight than the DKD rats (p < 0.05). Although the AS-IV treatment group exhibited a consistent rise in weight over the initial 6 weeks compared to the model group, the body weights of all DKD groups remained almost unchanged thereafter.

As shown in Figure 1B, the FBG levels in DKD rats were considerably elevated compared to the control group (p < 0.01). However, after 8 weeks of AS-IV administration, a significant decrease in FBG levels was observed in comparison to the model group (p < 0.05).

Compared to the control group, the levels of TG, TC, and LDL-C showed a significant increase in DKD rats (Figures 1C–E, p < 0.01). However, upon administering AS-IV at doses of 40 and 80 µM for a duration of 12 weeks, a substantial decrease in their levels was observed (p < 0.01).

As shown in Figures 2A–E, DKD rats had elevated levels of 24-h urine volume, urinary total protein, albumin-to-creatinine ratio (ACR), BUN, and Scr compared to the control group. Nevertheless, administration of AS-IV led to a decrease in 24-h urine volume, urinary total protein, and ACR in DKD rats (Figures 2A–C, p < 0.05). The BUN and Scr levels showed a decline after AS-IV treatment in comparison to the model group, but the difference was not statistically significant (Figures 2D, E, p > 0.05).

In order to confirm the impact of AS-IV on kidney damage, kidney tissue was subjected to H&E, PAS, and Masson staining. In comparison to the control group, rats in the DKD model group displayed kidney damage, which included enlarged glomeruli, noticeable growth of mesangial cells, elevated deposition of extracellular matrix (ECM), fibrosis in the interstitium, swelling of tubular epithelial cells, vacuolar degeneration, and loss of brush border (Figure 3A). Nevertheless, these anomalous alterations were mitigated in both the AS-IV treatment groups and the atorvastatin treatment group. Furthermore, a notable improvement was observed in the groups that received 40 and 80 µM AS-IV.

Mori et al. found that renal tubular cells take up fatty acids through Kidney Injury Molecule-1 (KIM-1), which leads to tubule injury accompanied by interstitial inflammation and fibrosis in DN (Mori et al., 2021). Kidney injury molecule 1 (KIM-1), liver fatty acid-binding protein (L-FABP), and neutrophil gelatinase-associated lipocalin (NGAL) are urinary biomarkers of renal tubular injury (Fufaa et al., 2015). The protein levels of KIM-1, NGAL, and L-FABP were significantly increased in DKD rats compared to the control group, as shown by Western blot analysis (Figures 3B–E). Additionally, the protein levels of KIM-1, L-FABP, and NGAL were decreased in the groups that received 40 and 80 µM AS-IV treatment. Taken together, these results showed that AS-IV could alleviate renal tubular injury in DKD.

Figures 3F, G demonstrates a notable decrease in NLRP3 expression in both the AS-IV treatment groups and the atorvastatin treatment group, as indicated by immunofluorescence staining for NLRP3. Furthermore, this reduction was particularly pronounced in the 40 µM group.

PCR analysis was conducted to determine the mRNA levels of IL-1β, IL-6, TNF-α, and MCP1 in order to explore the protective mechanism of AS-IV against DKD. According to Figures 3H–K, the mRNA levels of IL-1β, IL-6, TNF-α, and MCP1 were notably increased in DKD rats compared to the control group (p < 0.01). Following a 12-week treatment with AS-IV, a significant reduction in the mRNA levels of these inflammatory indicators was noted in the kidney tissue (p < 0.01).

Furthermore, our study found that HIF-1a expression was significantly elevated in rats with DKD (Figure 3L), while the HIF-1a mRNA level was significantly decreased after treatment with AS-IV or atorvastatin (p < 0.01). Growing evidence indicated that the effect of hypoxia and hypoxia-inducible factor-1 (HIF-1α) on renal tubules plays a critical role in the progression of DKD (Sun et al., 2012; Jiang et al., 2020; Feng et al., 2021). Additionally, hypoxia and HIF-1α promote podocyte injury and contribute to proteinuria (Nakuluri et al., 2019a; Nakuluri et al., 2019b). This illustrated that the mechanism by which AS-IV alleviates renal injury in DKD may be related to improving renal hypoxia.

The protein levels of CD36, NLRP3, ASC, cleaved-Caspase-1, cleaved-IL-1β, cleaved-GSDMD-D, and GSDMD-D were analyzed to further elucidate the relationship between NLRP3 inflammasome activation and CD36 upregulation through western blot analysis using kidney tissue samples (Figures 4A–H). Our findings revealed that DKD rats exhibited significantly higher protein levels of CD36, NLRP3, ASC, cleaved-caspase-1, cleaved-IL-1β, cleaved-GSDMD-D, and GSDMD-D compared to the control group. However, the expression of these proteins was significantly decreased after treatment with AS-IV or atorvastatin (p < 0.01).

The CCK-8 assay was used to assess the effect of AS-IV on the cytotoxicity of PA in HK-2 cells. Following a 24-h exposure to PA, there was a notable decline in cell viability, which was restored by AS-IV at concentrations of 40 and 80 µM (p < 0.01). Nevertheless, treatment with 20 µM AS-IV did not have a noteworthy impact on cell viability (p > 0.05). These findings suggest that PA can induce cell death in HK-2 cells and this effect can be alleviated with AS-IV at proper concentrations (Figure 5A).

Furthermore, PCR and western blot were utilized to assess the mRNA and protein levels of NLRP3, ASC, Caspase-1, and IL-1β in order to investigate the impact of AS-IV treatment on PA-induced HK-2 cells. As depicted in Figures 5B–F, the mRNA levels of NLRP3, ASC, Caspase-1, IL-1β, and HIF-1a in PA-induced HK-2 cells were significantly higher compared to the control group (p < 0.01). However, treatment with AS-IV at concentrations of 40 and 80 µM resulted in a significant decrease in these levels (p < 0.01). Additionally, the protein levels of NLRP3, ASC, cleaved-Caspase-1, cleaved-IL-1β, cleaved-GSDMD-D, GSDMD, KIM-1, NGAL, and L-FABP were evaluated. As shown in Figures 5G–P, the protein levels of these factors were elevated compared to the control group. However, they were notably diminished after AS-IV treatment (p < 0.01).

After treatment with AS-IV, the immunofluorescence staining for NLRP3 in HK-2 cells was significantly improved, as shown in Figures 5Q, R. The results indicate that the activation of the NLRP3 inflammasome is linked to cell demise caused by PA, whereas AS-IV has the ability to block this.

Previous research has demonstrated that CD36 potentially assumes a pivotal function in metabolic kidney disorders, while the accumulation of lipids facilitated by CD36 may be linked to activation of the NLRP3 inflammasome. In order to further examine the mechanism by which AS-IV suppresses the activation of the NLRP3 inflammasome, we measured the levels of CD36 through quantitative RT-PCR and western blot analysis. As illustrated in Figures 6B, E, F, the levels of CD36 were significantly higher in the PA groups compared to the control group (p < 0.01), but they were significantly decreased after AS-IV treatment. CD36 is a crucial molecule involved in lipid transport. To confirm the association between CD36 and lipid metabolism, we investigated the expression levels of CPT1 and PPAR-α, along with ROS production. Additionally, intracellular neutral lipid accumulation was assessed using Oil Red O staining. Exposure to PA resulted in increased levels of lipid ROS and intracellular lipid accumulation, simultaneously reducing the expression of CPT1 and PPAR-α (Figures 6C, D, G, H, p < 0.01). However, these effects were significantly reversed following AS-IV treatment. The results suggest that PA-stimulated HK-2 cells exhibit elevated levels of both lipid ROS and CD36; however, AS-IV effectively attenuated CD36 expression and inhibited lipid ROS production. Recent studies (Sheedy et al., 2013; Dai et al., 2017) have demonstrated that CD36 triggers the NLRP3 inflammasome in atherosclerosis, while inhibition of CD36 suppresses NLRP3 inflammasome activation (Kang et al., 2016). Taken together, we hypothesize that AS-IV might alleviate the activation of the NLRP3 inflammasome by suppressing the expression of CD36 in PA-induced HK-2 cells.

To further confirm the protective impact of AS-IV through its inhibition of CD36, we conducted a rescue experiment by introducing a CD36 lentiviral vectors (adenovirus) to enhance CD36 expression in PA-induced HK-2 cells. CD36 overexpression diminished the suppressive impact of AS-IV on intracellular lipid buildup and ROS (Figures 7S, T, p < 0.01). Additionally, compared with other groups, the expression levels of CD36, NLRP3, ACS, Caspase-1, and IL-1β were significantly elevated in the CD36 overexpression groups. Notably, AS-IV treatment effectively suppressed the PA-induced upregulation of CD36, NLRP3, ASC, Caspase-1, and IL-1β; however, these inhibitory effects were reversed upon CD36 overexpression (Figures 7C–R, p < 0.01). Collectively, these findings indicate that AS-IV mitigates PA-induced HK-2 cell injury through inhibiting CD36 expression.