Human proximal tubular cells (HK-2 cells) were cultured in DMEM/F-12 medium supplemented with 10% fetal bovine serum (FBS) in a humidified incubator containing 5% CO₂ at 37°C. Transient transfection of HK-2 cells with small interfering RNA (siRNA) was conducted with Lipofectamine 2000 (Invitrogen, CA, USA), and siRNAs (Nrf2 siRNA F: 5′-GCCCAUUGAUGUUUCUGAUTT-3′, R: 5′-AUCAGAAACGAAAUCAAUGGGCTT-3′; scrambled siRNA F: 5′-UUCUCCGAACGUGUCACGUTT-3′, R: 5′-ACGUGACACGUUCGGAGAATT-3′) were synthesized by GenePharma Company (Shanghai, China). For the overexpression of Nrf2, cells were transfected with Nrf2-pcDNA3.1-3xflag-C plasmids (Fenghbio, Hunan, China) using Lipofectamine 2000 reagent. MCC950 (10 μM), Mito Tempol (100 μM), or tBHQ (30 μM) was added to the culture medium for 2 h before exposure to palmitic acid (PA) media.

Male Sprague−Dawley rats (n = 24, aged 4 weeks) were obtained from the Animal Center of Chongqing Medical University and housed under pathogen-free conditions with free access to food and water. The rats were divided randomly into the following three groups: a control group, a high-fat diet (HFD) group, and a HFD group treated with tBHQ. The control group rats were fed a normal chow diet (#D12450B; 10 kcal% fat; Research Diet), and the HFD group was fed a Western high-fat diet (#D12451; 45 kcal% fat; Research Diet). The HFD-fed rats in the treatment group were intraperitoneally injected with tBHQ (50 mg/kg, every other day for 12 weeks; Sigma−Aldrich, USA), whereas the control group rats were injected with an equal volume of normal saline. All groups of rats were sacrificed at 20 weeks of age, and blood and kidney tissue samples were obtained for further experimental analysis. All animal experiments were performed in strict accordance with the Ethics Committee of Chongqing Medical University.

Kidney tissue samples were collected from the Pathology Department of the First Affiliated Hospital of Chongqing Medical University. For the control group, kidney tissues were collected from six patients who underwent nephrectomy for renal trauma with no other evidence of renal disease. Kidney tissues were collected from eight CKD patients with hyperlipidemia who underwent renal biopsy. Informed consent was obtained from all patients, and the experiments were approved by the Ethics Committee of The First Affiliated Hospital of Chongqing Medical University. Nrf2 and NLRP3 expression was measured by immunofluorescence and immunohistochemistry.

To measure mitochondrial ROS, HK-2 cells were seeded on slides in 12-well culture plates. After treatment, live HK-2 cells were stained with 10 μM DCFH-DA and 50 nM MitoTracker Red for 30 min at 37°C or incubated with MitoSOX™ Red fluorescent probe (5 μM, M36008, Invitrogen, USA) at 37°C for 20 min. Moreover, 4-μm-thick frozen sections were stained with 5 μM MitoSOX™ Red fluorescent probe for 20 min at 37°C in the dark. After being washed three times with phosphate-buffered saline (PBS), the stained HK-2 cells and frozen kidney sections were immediately visualized under a fluorescence microscope. The mitochondrial membrane potential was evaluated by using JC-1 dye (Beyotime, China), and live HK-2 cells or frozen kidney sections were incubated with JC-1 staining solution for 20 min at 37°C in the dark. After the cells were washed three times with PBS, images were subsequently acquired with a fluorescence microscope.

Paraffin-embedded renal tissue samples were sliced into 4-mm-thick sections. After deparaffinization in xylene and rehydration in a series of graded alcohols, the target antigens of the tissues were retrieved by microwaving for 15 min in 10 mmol/L sodium citrate buffer (pH 6.0), and endogenous peroxidases were inactivated by 0.3% H₂O₂ for 15 min at room temperature. After being washed, the sections were blocked in normal goat serum buffer for 15 min and then incubated with the following primary antibodies at 4°C overnight: rabbit anti-Nrf2 (1:200, Abcam), rabbit anti-HO-1 (1:200, Abcam), rabbit anti-NQO-1 (1:200, Abcam), rabbit anti-NLRP3 (1:200, CST), rabbit anti-caspase1 (1:200, CST), rabbit anti-IL-18 (1:200, Abcam), and mouse anti-8-OHdG (1:200, Santa Cruz). The sections were washed with PBS, incubated with a biotinylated secondary antibody (Zhongshan Golden Bridge Inc., China) for 30 min, incubated using a 3,3′-diaminobenzidine (DAB) kit for 3–5 min, counterstained with hematoxylin, dehydrated in ethanol, and visualized using a Nikon Eclipse 80i microscope. Quantification of the stained distribution area (area) and the integrated optical density (IOD) of the target protein in each IHC image was performed using ImageJ software. The average optical density (AOD = IOD/area) was used in this study for statistical analysis.

HK-2 cells were seeded on slides in 12-well culture plates. After various treatments, the cells were stained with MitoTracker Red at 37°C for 30 min in the dark, fixed with 4% paraformaldehyde for 10 min, and permeabilized in 0.1% Triton X-100 for 15 min at room temperature. After blocking with 5% BSA buffer, the cells were subsequently incubated with the indicated primary antibodies against NLRP3 (1:200), 8-OHdG (1:200), or Nrf2 (1:200) at 4°C overnight. After washing three times with PBS, the cells were incubated with the corresponding FITC-conjugated secondary antibodies and stained with DAPI. The images were captured using a fluorescence microscope.

Proteins from kidney tissues or HK-2 cells were electrophoretically separated on 8%–10% SDS−polyacrylamide gels and then transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% nonfat milk and incubated overnight at 4°C with the following primary antibodies: rabbit anti-NLRP3 (1:1,000, CST), rabbit anti-caspase1 (1:1,000, CST), rabbit anti-IL-18 (1:1,000, Abcam), rabbit anti-Nrf2 (1:1,000, Abcam), rabbit anti-HO-1 (1:5,000, Abcam), rabbit anti-NQO-1 (1:5,000, Abcam), rabbit anti-cleaved-caspase3 (1:1,000, CST), and mouse anti-β-actin (1:5,000, Sungene Biotech). Finally, the membranes were washed three times with TBST and then incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h, after which the protein bands were visualized using an electrochemiluminescence detection system (GE Healthcare, Piscataway, NJ, USA).

Cell apoptosis was detected using an Annexin V-FITC/PI apoptosis assay kit (Sungene Biotech, China). In brief, after double-staining with annexin V and propidine iodide (PI), the number of apoptotic cells was measured by flow cytometry analysis. To assess the extent of renal apoptosis, terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) staining (In Situ Cell Death Detection Kit, POD, Roche) was performed on frozen kidney sections, and the number of TUNEL-positive cells per 0.25 mm² was calculated.

All data are expressed as means ± SEM. Qualitative data were analyzed using Student’s t-test for comparisons between two groups or one-way analysis of variance (ANOVA) followed by Tukey’s posttest for comparisons among multiple groups. A value of P <0.05 was considered to indicate statistical significance.

To evaluate the activation of the NLRP3 inflammasome in PA-induced tubular epithelial cells, we determined the expression levels of NLRP3, mature caspase-1, and the proinflammatory cytokine IL-18, which are used as markers of inflammasome activation. As shown in Figures 1A–H , the protein expression of NLRP3, mature caspase-1, and IL-18 was significantly upregulated in HK-2 cells following PA stimulation in a dose- and time-dependent manner, as determined by western blot analyses. Moreover, immunofluorescence analysis revealed that PA induced an increase in NLRP3 protein expression compared to that in control cells ( Figure 1I ).

Next, we detected the expression of NLRP3 in the kidney tissue of CKD patients with hyperlipidemia. First, H&E and PAS staining revealed marked glomerular mesangial expansion and increased mesangial matrix in the kidneys of CKD patients with hyperlipidemia ( Figure 1J ). Masson’s trichrome staining revealed that renal fibrosis was enhanced in the kidneys of CKD patients with hyperlipidemia ( Figure 1J ). Subsequently, immunofluorescence analysis and immunohistochemical staining revealed that the expression of NLRP3 was significantly upregulated in the kidneys of CKD patients with hyperlipidemia ( Figures 1K–N ). Finally, we investigated the role of NLRP3 inflammasome activation in PA-induced HK-2 cell apoptosis. Flow cytometry showed that MCC950 (a NLRP3 antagonist) treatment significantly ameliorated PA-induced cell apoptosis ( Figures 1O, P ). Taken together, these results indicated that high lipid levels induced NLRP3 inflammasome activation, which promoted tubular epithelial cell apoptosis.

Mitochondria are major sources of ROS and are also targets of ROS. To investigate the role of mtROS in mitochondrial damage, HK-2 cells were treated with the mitochondrion-targeted antioxidant Mito Tempol. Immunofluorescence analysis revealed that Mito Tempol treatment significantly attenuated the production of mtROS ( Figures 2A, E ) and reversed the loss of (the mitochondrial membrane potential) ΔΨm in HK-2 cells under PA stimulation ( Figures 2B, F ). Nuclear and mitochondrial DNA oxidative damage can be assessed by immunofluorescence staining for nuclear and nonnuclear 8-OHdG, respectively (20). As shown in Figure 2C , immunofluorescence analysis demonstrated that PA induced an increase in the colocalization of 8-OHdG and mitochondria or nuclei, suggesting that PA induced mitochondrial and nuclear DNA oxidative damage in HK-2 cells. However, Mito Tempol treatment significantly ameliorated PA-induced oxidative damage to mitochondrial and nuclear DNA in HK-2 cells.

Mitochondrial damage plays a pivotal role in NLRP3 inflammasome activation, and previous studies revealed that mtROS generated by damaged mitochondria are critically involved in NLRP3 inflammasome activation (21). Similarly, our Western blot analysis showed that Mito Tempol treatment significantly prevented the PA-induced increase in the expression of NLRP3, caspase-1, and IL-18 ( Figures 2D, H–J ). As mitochondria can serve as a molecular platform for the assembly of NLRP3 inflammasome components (22, 23), which subsequently mediate the maturation and secretion of proinflammatory cytokines, we explored the subcellular localization of NLRP3 inflammasome components under PA stimulation conditions and found that NLRP3 was located in the cytoplasm but not within mitochondria in control cells. However, stimulation with PA led to the recruitment of NLRP3 within mitochondria, as demonstrated by the increasing overlap of NLRP3 in the mitochondria ( Figure 2G ). However, Mito Tempol treatment inhibited PA-induced NLRP3 upregulation and recruitment within mitochondria ( Figure 2G ). These results suggested that PA-induced NLRP3 inflammasome activation was dependent on mitochondrial ROS generation.

To determine the effects of PA exposure on the Nrf2/ARE signaling pathway, we measured the expression of Nrf2, HO-1, and NQQ-1 in HK-2 cells and found that they were noticeably increased in a dose-dependent manner under PA stimulation ( Figure 3A–D ). Additionally, we observed the expression and location of Nrf2 in HK-2 cells. Immunostaining revealed that PA treatment increased the Nrf2 fluorescence intensity and increased the colocalization of Nrf2 with the nucleus, indicating that Nrf2 translocated from the cytoplasm to the nucleus ( Figures 3E, F ). Furthermore, we detected the expression of Nrf2 in the kidney tissue of CKD patients with hyperlipidemia. As shown in Figures 3G–J , immunohistochemical staining and immunofluorescence analysis revealed that the expression of Nrf2 was significantly upregulated in the kidneys of CKD patients with hyperlipidemia. These results demonstrated that high lipid concentrations activate the Nrf2/ARE signaling pathway in tubular epithelial cells.

To determine the role of the Nrf2/ARE signaling pathway in mtROS production and mitochondrial function in PA-treated HK-2 cells, siRNA was used to silence Nrf2. Immunoblot analysis indicated that Nrf2 siRNA successfully suppressed the protein expression of Nrf2 ( Figures 4A, B ), and Nrf2 silencing significantly decreased the protein expression of Nrf2, HO-1, and NQO-1 in HK-2 cells treated with or without PA ( Figures 4C–F ). In addition, immunofluorescence staining showed that silencing Nrf2 significantly exacerbated mtROS production ( Figures 4G, H ) and the loss of ΔΨm ( Figures 4I, J ) in HK-2 cells cultured in PA media. These data suggested that inhibiting the Nrf2/ARE signaling pathway exacerbated PA-induced mtROS production and mitochondrial damage in HK-2 cells.

We next investigated the effect of tBHQ, a well-characterized Nrf2 activator, on the Nrf2 signaling pathway in HK-2 cells. HK-2 cells were treated with tBHQ at different concentrations (0–50 μM), and as expected, tBHQ dose-dependently increased the protein level of Nrf2 ( Figures 5A–D ). Consistently, the protein levels of Nrf2 downstream targets, including HO-1 and NQQ-1, were increased in response to tBHQ ( Figures 5A–D ). Next, we incubated HK-2 cells with PA in the absence or presence of 30 μM tBHQ for 24 h and found that tBHQ ameliorated PA-induced mitochondrial ROS production ( Figures 5F, G ), mitochondrial injury ( Figures 5H, I ), and mitochondrial DNA oxidative damage ( Figure 5J ). Similarly, transfection of HK-2 cells with an Nrf2 overexpression plasmid ( Figure 5E ) significantly attenuated mitochondrial ROS production ( Figures 5F, G ) and reversed PA-induced mitochondrial membrane potential collapse ( Figures 5H, I ) and mitochondrial DNA oxidative damage ( Figure 5J ).

To further investigate the possible role of Nrf2 in NLRP3 inflammasome activation, PA-stimulated HK-2 cells were pretreated with tBHQ, as shown in Figures 6A, F–H . tBHQ treatment markedly inhibited the activation of the NLRP3 inflammasome in PA-treated HK-2 cells, as characterized by decreased NLRP3, caspase-1. and IL-18 levels. Moreover, we found that tBHQ significantly inhibited PA-induced colocalization of NLRP3 and MitoTracker ( Figure 6B ) and ameliorated PA-induced cell apoptosis in HK-2 cells ( Figures 6C, D ), demonstrating that the Nrf2/ARE signaling pathway plays a protective role in PA-induced cell apoptosis by inhibiting NLRP3 inflammasome activation in HK-2 cells. Next, we studied the underlying mechanism of the inhibitory effects of the Nrf2/ARE signaling pathway on NLRP3 inflammasome activation. Considering the regulatory effect of the Nrf2/ARE pathway on mtROS production, HK-2 cells were pretreated with Mito Tempol or/and Nrf2 siRNA, and knockdown of Nrf2 significantly increased the PA-induced expression of NLRP3, mature caspase-1, and IL-18 ( Figure 6E ). However, the enhancing effect of Nrf2 siRNA on NLRP3 inflammasome activation was markedly reversed by Mito Tempol intervention ( Figures 6E, I–K ). Collectively, these results indicated that Nrf2-induced NLRP3 inflammasome inhibition was dependent on mtROS production.

As shown in Figures 7A–F , compared with those in LFD-fed rats, body weight, blood urea nitrogen (BUN), serum creatinine (SCr), urine protein, total cholesterol (TC), and triglyceride (TG) levels were significantly greater in HFD-fed rats; however, tBHQ intervention significantly attenuated these parameters, except for body weight, relative to those in HFD-fed rats. Oil red O staining showed that abnormal lipid accumulation was notably increased in renal proximal tubule cells of HFD-fed rats; however, tBHQ treatment markedly attenuated lipid deposition in renal proximal tubule cells in HFD-fed rats ( Figure 7G ). Additionally, H&E and PAS staining of the kidneys of HFD-fed rats revealed enhanced glomerular hypertrophy, mesangial proliferation, mesangial matrix deposition, and notable disruption of tubular epithelial cells compared to those of LFD-fed rats ( Figures 7H, I ). Masson trichrome staining revealed marked renal fibrosis in HFD-fed rats ( Figure 7J ); however, these changes were dramatically limited by tBHQ treatment. Moreover, mtROS generation ( Figures 7K, O ), ΔΨm loss ( Figures 7L, P ), and DNA oxidative damage ( Figures 7M, Q ) were enhanced in the kidneys of HFD-fed rats but markedly attenuated after tBHQ treatment. Furthermore, TUNEL staining demonstrated that the number of apoptotic cells (indicated by the arrow) was significantly reversed by tBHQ treatment ( Figures 7N, R ). Overall, these data demonstrated that tBHQ improved renal function in HFD-induced obese rats.

Finally, we investigated the role of the Nrf2/ARE pathway in the activation of the NLRP3 inflammasome in HFD-induced obese rats. Immunoblot analysis demonstrated that the expression of Nrf2, HO-1, NQO-1, NLRP3, mature-caspase-1, and IL-18 was significantly upregulated in the HFD group compared to the LFD group ( Figures 8A, C ). Additionally, the expression of Nrf2, HO-1, and NQQ-1 was significantly greater in HFD-fed rats after tBHQ intervention ( Figures 8A, C ). However, the HFD-induced increase in the expression of NLRP3, mature caspase-1, and IL-18 was significantly attenuated following treatment with tBHQ ( Figures 8A,C ). Subsequently, immunohistochemical staining ( Figures 8B, D ) also supported the above-mentioned results, indicating that the upregulation of the Nrf2/ARE pathway inhibited the activation of the NLRP3 inflammasome in the kidneys of HFD-induced obese rats.